Soft anticholinergic esters

ABSTRACT

Soft anticholinergic esters of the formulas: 
                         
wherein R 1  and R 2  are both phenyl or one of R 1  and R 2  is phenyl and the other is cyclopentyl; R is C 1 -C 8  alkyl, straight or branched chain; and X −  is an anion with a single negative charge; and wherein each asterisk marks a chiral center; said compound having the R, S or RS stereoisomeric configuration at each chiral center unless specified otherwise, or being a mixture thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/494,367, filed Jun. 30, 2009, now U.S. Pat. No. 8,147,809, which is adivisional of U.S. patent application Ser. No. 12/138,013, filed Jun.12, 2008, now U.S. Pat. No. 7,576,210, which is a divisional of U.S.patent application Ser. No. 11/598,079, filed Nov. 13, 2006, now U.S.Pat. No. 7,399,861, which claims benefit of U.S. Provisional PatentApplication No. 60/735,207, filed Nov. 10, 2005, all incorporated byreference herein in their entireties and relied upon.

This application is also related to U.S. application Ser. No. 11/598,076concurrently filed with prior application Ser. No. 11/598,079 on Nov.13, 2006, by the present inventor and claiming benefit of U.S.Provisional Application No. 60/735,206, filed Nov. 10, 2006, now U.S.Pat. No. 7,417,174, as well as application Ser. No. 12/137,896, filedJun. 12, 2008, as a divisional of application Ser. No. 11/598,076, nowU.S. Pat. No. 7,538,219, and its divisional, application Ser. No.12/418,939, filed Apr. 6, 2009, now U.S. Pat. No. 8,071,639, as well asthe divisional thereof, application Ser. No. 13/286,020, filed Oct. 31,2011, all incorporated by reference herein in their entireties andrelied upon.

BACKGROUND

Various anticholinergic compounds have been previously described but arenot optimal.

Muscarinic receptor antagonists are frequently used therapeutic agentsthat inhibit the effects of acetylcholine by blocking its binding tomuscarinic cholinergic receptors at neuroeffector sites on smoothmuscle, cardiac muscle, and gland cells as well as in peripherialganglia and in the central nervous system (CNS). However, their sideeffects, which can include dry mouth, photophobia, blurred vision,urinary hesitancy and retention, decreased sweating, drowsiness,dizziness, restlessness, irritability, disorientation, hallucinations,tachycardia and cardiac arrhythmias, nausea, constipation, and severeallergic reactions, often limit their clinical use, and even topicalanticholinergics can cause the same unwanted side effects.Glycopyrrolate and triotropium are among the quaternary ammoniumanticholinergics, which have reduced CNS-related side effects as theycannot cross the blood-brain barrier, however, because glycopyrrolate(or, presumably, tiotropium) is eliminated mainly as unchanged drug oractive metabolite in the urine, its administration is problematic inyoung or elderly patients and especially in uraemic patients. Toincrease the therapeutic index of anticholinergics, the soft drugapproach has been applied in a number of different designs starting fromvarious lead compounds over the past 20 years, but there is a need foryet other new soft anticholinergics. These novel muscarinic antagonists,just as all other soft drugs, are designed to elicit their intendedpharmacological effect at the site of application, but to be quicklymetabolized into their designed-in, inactive metabolite upon enteringthe systemic circulation and rapidly eliminated from the body, resultingin reduced systemic side effects and increased therapeutic index.

SUMMARY

New soft anticholinergic agents, pharmaceutical compositions containingthem, processes for their preparation and methods for eliciting ananticholinergic response, especially for treating an obstructive diseaseof the respiratory tract or for treating overactive bladder, areprovided.

In one exemplary embodiment, there is provided a compound having theformula

wherein R₁ and R₂ are both phenyl or one of R₁ and R₂ is phenyl and theother is cyclopentyl; R is C₁-C₈ alkyl, straight or branched chain; andX⁻ is an anion with a single negative charge; and wherein each asteriskmarks a chiral center; said compound having the R, S or RSstereoisomeric configuration at each chiral center unless otherwisespecified, or being a mixture thereof.

In another exemplary embodiment, there is provided a compound having theformula

wherein R is methyl or ethyl.

In other exemplary embodiments, processes for preparing the compoundsare provided.

In other exemplary embodiments, there arm provided pharmaceuticalcompositions comprising one or more of the compounds of the foregoingformulas and pharmaceutically acceptable carriers therefor;pharmaceutical combinations comprising one or more of the compounds ofthe foregoing formulas and an anti-inflammatory corticosteroid, abetamimetic agent or an antialleric agent; and methods of using thesubject compositions and combinations.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the pH profiles of four compounds of the invention:Compound (a), -◯-; Compound (b), -Δ-; Compound (c), -▴-; and Compound(d), -•-.

FIG. 2 is a representative time-profile of a chemical hydrolysis withformation of the corresponding acid as hydrolytic product [Compound (d),pH 7.3, 37° C.].

FIG. 3A is a graph of mydriatic activities of glycopyrrolate and softanalogs Compound (c) and Compound (d) at pharmacologically equipotentdoses (mean±SD shown) showing data for up to 144 hours.

FIG. 3B is a graph of mydriatic activities of glycopyrrolate and softanalogs Compounds (c) and (d) at pharmacologically equipotent doses(mean±SD shown) showing data for the first 24 hours only.

FIG. 4 is a graph of mydriatic activities of various zwitterionicisomers at 0.1% concentrations over a seven hour period.

FIG. 5 is a graph comparing the mydriatic activity of the most activezwitterionic isomers with glycopyrrolate at 0.1% concentrations.

FIG. 6 is a graph of the heart rate in beats per minute versus time inminutes showing the protection effect of different anticholinergics oncarbachol-induced bradycardia in anesthetized rats (mean±SD; n=3-5),including ethyl, n-hexyl and n-octyl esters.

FIG. 7 is a graph of the heart rate in beats per minute versus time inminutes showing the protective effect of different anticholinergics,including Compound (w), on carbachol-induced bradycardia is anesthetizedrats (n=3-6).

FIG. 8 is a graph showing the time course of action of differentanticholinergics, including Compounds (w) and (aa), on electricallystimulated guinea pig trachea.

FIG. 9 is a graph showing the time course of the effect of differentanticholingerics including Compounds (w) and (aa), after wash out of thetest drug on electrically stimulated guinea pig trachea.

FIG. 10 is a graph of bronchoconstriction (% of baseline) versus timefor acetylcholine-induced bronchoconstriction in anesthetized guineapigs for Compounds (q) and (m) and glycopyrrolate at selected dosages.

DETAILED DESCRIPTION

Throughout this specification, the following definitions, generalstatements and illustrations are applicable:

The patents, published applications, and scientific literature referredto herein establish the knowledge of those with skill in the art and arehereby incorporated by reference in their entirety to the same extent asif each was specifically and individually indicated to be incorporatedby reference. Any conflict between any reference cited herein and thespecific teachings of this specification shall be resolved in favor ofthe latter. Likewise, any conflict between an art-understood definitionof a word or phrase and a definition of the word or phrase asspecifically taught in this specification shall be resolved in favor ofthe latter.

As used herein, whether in a transitional phrase or in the body of aclaim, the terms “comprise(s)” and “comprising” are to be interpreted ashaving an open-ended meaning. That is, the terms are to be interpretedsynonymously with the phrases “having at least” or “including at least”.When used in the context of a process, the term “comprising” means thatthe process includes at least the recited steps, but may includeadditional steps. When used in the context of a composition, the term“comprising” means that the composition includes at least the recitedfeatures or components, but may also include additional features orcomponents.

The terms “consists essentially of” or “consisting essentially of” havea partially closed meaning, that is, they do not permit inclusion ofsteps or features or components which would substantially change theessential characteristics of a process or composition; for example,steps or features or components which would significantly interfere withthe desired properties of the compounds or compositions describedherein, i.e., the process or composition is limited to the specifiedsteps or materials and those which do not materially affect the basicand novel characteristics of the invention. The basic and novel featuresherein are the provision of compounds of formula (Ia) and (Ib) andcombinations of those compounds with other drugs, particularly withanti-inflammatory steroids, especially loteprednol etabonate oretiprednol dichloracetate, and most especially in the case of loteprenoletabonate (LE) further including an inactive metabolite enhancing agentfor the LE as further defined hereinafter.

The terms “consists of” and “consists” are closed terminology and allowonly for the inclusion of the recited steps or features or components.

As used herein, the singular forms “a,” “an” and “the” specifically alsoencompass the plural forms of the terms to which they refer, unless thecontent clearly dictates otherwise.

The term “about” is used herein to mean approximately, in the region of,roughly, or around. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” or “approximately” is used herein to modify a numerical valueabove and below the stated value by a variance of 20%.

As used herein, the recitation of a numerical range for a variable isintended to convey that the variable can be equal to any of the valueswithin that range. Thus, for a variable which is inherently discrete,the variable can be equal to any integer value of the numerical range,including the end-points of the range. Similarly, for a variable whichis inherently continuous, the variable can be equal to any real value ofthe numerical range, including the end-points of the range. As anexample, a variable which is described as having values between 0 and 2,can be 0, 1 or 2 for variables which are inherently discrete, and can be0.0, 0.1, 0.01, 0.001, or any other real value for variables which areinherently continuous.

In the specification and claims, the singular forms include pluralreferents unless the context clearly dictates otherwise. As used herein,unless specifically indicated otherwise, the word “or” is used in the“inclusive” sense of “and/or” and not the “exclusive” sense of“either/or.”

Technical and scientific terms used herein have the meaning commonlyunderstood by one of skill in the art to which the present inventionpertains, unless otherwise defined. Reference is made herein to variousmethodologies and materials known to those of skill in the art. Standardreference works setting forth the general principles of pharmacologyinclude Goodman and Gilman's The Pharmacological Basis of Therapeutics,10^(th) Ed., McGraw Hill Companies Inc., New York (2001).

As used herein, “treating” means reducing, preventing, hindering orinhibiting the development of, controlling, alleviating and/or reversingthe symptoms in the individual to which a combination or compositioncomprising a compound of formula (Ia) or (Ib) has been administered, ascompared to the symptoms of an individual not being so treated. Apractitioner will appreciate that the combinations, compositions, dosageforms and methods described herein are to be used in concomitance withcontinuous clinical evaluations by a skilled practitioner (physician orveterinarian) to determine subsequent therapy. Such evaluation will aidand inform in evaluating whether to increase, reduce or continue aparticular treatment dose, and/or to alter the mode of administration.

The methods described herein are intended for use with anysubject/patient that may experience their benefits. Thus, the terms“subjects” as well as “patients,” “individuals” and “warm-bloodedanimals” include humans as well as non-human subjects, particularlydomesticated animals, particularly dogs, cats, horses and cows, as wellas other farm animals, zoo animals and/or endangered species.

X⁻ denotes an anion with a single negative charge. This anion is ananion of a pharmaceutically acceptable acid. Preferably. X⁻ is chloride,bromide, iodide, sulfate, phosphate, methanesulfonate, nitrate, maleate,acetate, citrate, fumarate, tartrate, oxalate, succinate, benzoate orp-toluenesulfonate. More preferably, X⁻ is chloride, bromide,4-toluenesulfonate or methanesulfonate. Most preferably X⁻ is bromide.

In formula (Ia), the compounds having the R configuration with respectto chiral center 2 are of particular interest.

The moiety R in formulas (Ia) and (Ib) can be methyl, ethyl, n-propyl,n-butyl, n-pentyl, n-hexyl, n-heptyl or n-octyl or their branched chainisomers.

In the compounds of formulas (Ia) and (Ib), R is preferably C₁-C₆straight chain alkyl.

In the compounds of formula (Ia), compounds wherein one of R₁ and R₂ isphenyl and the other is cyclopentyl are of particular interest.

Also of particular interest are the compounds of the formula:

wherein R is methyl or ethyl.

The following specific compounds of formula (Ia) are of particularinterest:

-   (a)    3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidinium    bromide;-   (b)    3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidinium    bromide;-   (c) (2R)    3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidinium    bromide;-   (d) (2R)    3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidinium    bromide;-   (e) (2R,3′R)    3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidinium    bromide;-   (f) (2R,3′S)    3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidinium    bromide;-   (g) (2R,3′S)    3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidinium    bromide;-   (h) (2S,3′R)    3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidinium    bromide;-   (i) (2S,3′S)    3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidinium    bromide;-   (j) (2R,3′S)    3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidinium    bromide;-   (k) (2R,1′R, 3′S)    3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidinium    bromide;-   (l) (2R,1′S,3′S)    3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidinium    bromide;-   (m) (2R,1′R,3′R)    3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidinium    bromide;-   (n) (2R,1′S,3′R)    3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidinium    bromide;-   (o) (2R,1′R,3′S)    3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(n-hexyloxycarbonylmethyl)-1-methylpyrrolidinium    bromide;-   (p) (2R,1′S,3′S)    3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(n-hexyloxycarbonylmethyl)-1-methylpyrrolidinium    bromide;-   (q) (2R,1′R, 3′R)    3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(n-hexyloxycarbonylmethyl)-1-methylpyrrolidinium    bromide;-   (r) (2R,1′S, 3′R)    3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(n-hexyloxycarbonylmethyl)-1-methylpyrrolidinium    bromide;-   (s) (2R,1′R, 3′S)    3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-methyl-1-(n-octyloxycarbonylmethyl)pyrrolidinium    bromide;-   (t) (2R,1′S,3′S)    3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-methyl-1-(n-octyloxycarbonylmethyl)pyrrolidinium    bromide;-   (u) (2R,1′R,3′R)    3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-methyl-1-(n-octyloxycarbonylmethyl)pyrrolidinium    bromide; or-   (v) (2R,1′S,3′R)    3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-methyl-1-(n-octyloxycarbonylmethyl)pyrrolidinium    bromide.

Of these, particular mention may be made of:

-   (a)    3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidinium    bromide;-   (b)    3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidinium    bromide;-   (c) (2R)    3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidinium    bromide; or-   (d) (2R)    3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidinium    bromide.-   In formula (Ib), the compound wherein R is ethyl and X⁻ is Br⁻ is of    special interest. The compounds of formula (Ib) wherein R is methyl,    n-hexyl and n-octyl and X⁻ is Br⁻ can be made in analogous fashion    to the R=ethyl compound and are also of particular interest.

Various methods of making the instant compounds are illustratedhereinafter. Generally speaking, the compounds of formula (Ia) can beprepared by reacting a bromoacetate of the formulaBrCH₂COORwherein R is as defined above, with a compound of the formula

wherein R₁ and R₂, the asterisks and the stereoisomeric configurationsare as defined above, and optionally separating the individualstereoisomers to afford a compound of formula (Ia) and, when desired,exchanging the bromine anion with a different X⁻ anion wherein X⁻ is asdefined above but other than Br⁻.

In a particular embodiment, the compound of formula (IIa) has the Rconfiguration with respect to chiral center 2.

In another particular embodiment, the compound of formula (IIa) has theconfiguration R or S with respect to chiral center 1′ or with respect tochiral center 3′.

In another embodiment, the process includes separating the individualstereoisomers of the compound of formula (Ia) after their formation tothe extent possible.

In one particular embodiment, the process comprises quaternmizing acompound of the formula

with an alkyl bromoacetate of the formulaBrCH₂COORwherein R is methyl or ethyl, to afford the desired product.

In analogous fashion, methods of making the compounds of formula (Ib)are illustrated hereinafter. Generally speaking, the process comprisesreacting a bromocetate of the formula:BrCH₂COORwherein R is as defined above, with a compound of the formula

and optionally separating the individual stereoisomers to afford acompound of formula (Ib) and, when desired, exchanging the bromine anionwith a different X⁻ anion wherein X⁻ is as defined above but other thanBr⁻.

In the case of both the compounds of formula (Ia) and those of formula(Ib), use of ICH₂COOR or ClCH₂COOR in place of BRCH₂COOR can be employedin the above reaction schemes to afford the corresponding compounds inwhich X⁻ is I⁻ or Cl⁻. Alternatively, ion exchange columns can be usedto replace the Br anion in the product of formula (Ia) or (Ib) with adifferent X⁻ anion.

The compounds of formulas (Ia) and (Ib) are of use as pharmaceuticalagents because of their anticholinergic activity. An anticholinergicallyeffective amount of such an agent inhibits the effect of acetycholine byblocking its binding to muscarinic cholinergic receptors atneuroeffector sites. Subjects in need of a method of eliciting ananticholinergic response are those suffering from conditions whichrespond to treatment with an anticholinergic agent. Such conditionsinclude obstructive diseases of the respiratory tract, for exampleasthma and chronic obstructive pulmonary disease, vagally induced sinusbradycardia and heart rhythm disorders, spasms, for example in thegastrointestinal tract or urinary tract (including overactive bladder)and in menstrual disorders. The compounds of formulas (Ia) and (Ib) canalso be used to induce short-acting mydriasis and thus can be used todilate the pupils of the eyes in vision testing. Other uses of thecompounds of formulas (Ia) and (Ib) include the treatment of ulcers aswell as topical use as an antiperspirant in the treatment hyperhydrosis(sweating).

The compounds of formula (Ia) and (Ib) are particularly useful in thetreatment of obstructive diseases of the respiratory tract. Theexpression “obstructive disease of the respiratory tract” includesbreathing disorders such as asthma, bronchitis, chronic obstructivepulmonary disease (COPD), allergic rhinitis and infectious rhinitis.

“Asthma” refers to a chronic lung disease causing bronchoconstriction(narrowing of the airways) due to inflammation (swelling) and tighteningof the muscles around the airways. The inflammation also causes anincrease in mucus production, which causes coughing that may continuefor extended periods. Asthma is generally characterized by recurrentepisodes of breathlessness, wheezing, coughing, and chest tightness,termed exacerbations. The severity of exacerbations can range from mildto life threatening. The exacerbations can be a result of exposure toe.g. respiratory infections, dust, mold, pollen, cold air, exercise,stress, tobacco smoke, and air pollutants.

“COPD” refers to chronic obstructive pulmonary disease, primarily butnot necessarily associated with past and present cigarette smoking. Itinvolves airflow obstruction, mainly associated with emphysema andchronic bronchitis. Emphysema causes irreversible lung damage byweakening and breaking the air sacs within the lungs. Chronic bronchitisis an inflammatory disease, which increases mucus in the airways andbacterial infections in the bronchial tubes, resulting in obstructedairflow.

“Allergic rhinitis” refers to acute rhinitis or nasal rhinitis,including hay fever. It is caused by allergens such as pollen or dust.It may produce sneezing, congestion, runny nose, and itchiness in thenose, throat, eyes, and ears.

“Infectious rhinitis” refers to acute rhinitis or nasal rhinitis ofinfectious origin. It is caused by upper respiratory tract infection byinfectious rhinoviruses, coronaviruses, influenza viruses, parainfluenzaviruses, respiratory syncytical virus, adenoviruses, coxsackieviruses,echoviruses, or Group A beta-hemolytic Streptococci and is genericallyreferred to as the common cold. It may produce sneezing, congestion,runny nose, and itchiness in the nose, throat, eyes, and ears.

The compounds of formula (Ia) and (Ib) are also particularly useful inthe treatment of overactive bladder (OAB).

Overactive bladder is a treatable medical condition that is estimated toaffect 17 to 20 million people in the United States. Symptoms ofoveractive bladder can include urinary frequency, urinary urgency,urinary urge incontinence (accidental loss of urine) due to a sudden andunstoppable need to urinate, nocturia (the disturbance of nighttimesleep because of the need to urinate) or enuresis resulting fromoveractivity of the detrusor muscle (the smooth muscle of the bladderwhich contracts and causes it to empty).

Neurogenic overactive bladder (or neurogenic bladder) is a type ofoveractive bladder which occurs as a result of detrusor muscleoveractivity referred to as detrusor hyperreflexia, secondary to knownneurologic disorders. Patients with neurologic disorders, such asstroke, Parkinson's disease, diabetes, multiple sclerosis, peripheralneuropathy, or spinal cord lesions often suffer from neurogenicoveractive bladder. In contrast, non-neurogenic overactive bladderoccurs as a result of detrusor muscle overactivity referred to asdetrusor muscle instability. Detrusor muscle instability can arise fromnon-neurological abnormalities, such as bladder stones, muscle disease,urinary tract infection or drug side effects or can be idiopathic.

Due to the enormous complexity of micturition (the act of urination), anexact mechanism which causes overactive bladder is not known. Overactivebladder can result from hypersensitivity of sensory neurons of theurinary bladder, arising from various factors including inflammatoryconditions, hormonal imbalances, and prostate hypertrophy. Destructionof the sensory nerve fibers, either from a crushing injury to the sacralregion of the spinal cord, or from a disease that causes damage to thedorsal root fibers as they enter the spinal cord can also lead tooveractive bladder. In addition, damage to the spinal cord or brain stemcausing interruption of transmitted signals can lead to abnormalities inmicturition. Therefore, both peripheral and central mechanisms can beinvolved in mediating the altered activity in overactive bladder.

Current treatments for overactive bladder include medication, dietmodification, programs in bladder training, electrical stimulation, andsurgery. Currently, antimuscarinics (which are members of the generalclass of anticholinergics) are the primary medication used for thetreatment of overactive bladder. The antimuscarinic, oxybutynin, hasbeen the mainstay of treatment for overactive bladder. However,treatment with known antimuscarinics suffers from limited efficacy andside effects such as dry mouth, dry eyes, dry vagina, blurred vision,cardiac side effects, such as palpitations and arrhythmia, drowsiness,urinary retention, weight gain, hypertension and constipation, whichhave proven difficult for some individuals to tolerate. Thus, the needfor new anticholinergic agents is evident.

The compounds of formula (Ia) or (Ib) may be used on their own orcombined with other active substances of formula (Ia) or (Ib) accordingto the invention.

The compounds of formula (Ia) or (Ib) may optionally also be combinedwith other pharmacologically active substances. These include, inparticular, betamimetics, antiallergic agents, and corticosteroids (alsotermed “anti-inflammatory steroids”, “anti-inflammatory corticosteroids”or simply “steroids”) and combinations of these active substances. Thecombinations with betamimetics, antiallergics or corticosteroids are ofinterest in the treatment of obstructive diseases of the respiratorytract, especially COPD or asthma. Accordingly, they are intended foradministration by oral inhalation, as powders or aerosols.

Examples of betamimetics which may be used in conjunction with thecompounds of formula (Ia) or (Ib) include compounds selected from thegroup consisting of bambuterol, bitolterol, carbuterol, clenbuterol,fenoterol, formoterol, hexoprenaline, ibuterol, pirbuterol, procaterol,reproterol, salmeterol, sulfphonterol, terbutaline, tulobuterol,4-hydroxy-7-[2-{[2-{[3-(2-phenylethoxy)propyl]sulfonyl}ethyl]amino}ethyl]-2(3H)-benzothiazolone,1-(2-fluoro-4-hydroxyphenyl)-2-[4-(1-benzimidazolyl)-2-methyl-2-butylamino]ethanol,1-[3-(4-methoxybenzylamino)-4-hydroxyphenyl]-2-[4-(1-benzimidazolyl)-2-methyl-2-butylamino]ethanol,1-[2H-5-hydroxy-3-oxo-4H-1,4-benzoxazin-8-yl]-2-[3-(4-N,N-dimethylaminophenyl)-2-methyl-2-propylamino]ethanol,1-[2H-5-hydroxy-3-oxo-4H-1,4-benzoxazin-8-yl]-2-[3-(4-methoxyphenyl)-2-methyl-2-propylamino]ethanol,1-[2H-5-hydroxy-3-oxo-4H-1,4-benzoxazin-8-yl]-2-[3-(4-n-butyloxyphenyl)-2-methyl-2-propylamino]ethanol,1-[2H-5-hydroxy-3-oxo-4H-1,4-benzoxazin-8-yl]-2-(4-[3-(4-methoxyphenyl)-1,2,4-triazol-3-yl]-2-methyl-2-butylaminoethanol,5-hydroxy-8-(1-hydroxy-2-isopropylaminobutyl)-2H-1,4-benzoxazin-3-(4H)-one,1-(4-amino-3-chloro-5-trifluoromethylphenyl)-2-tert.-butylamino)ethanoland1-(4-ethoxycarbonylamino-3-cyano-5-fluorophenyl)-2-(tert.-butylamino)ethanol,optionally in the form of their racemates, their enantiomers, theirdiastereomers, as well as optionally their pharmacologically acceptableacid addition salts and hydrates. It is particularly preferable to use,as betamimetics, active substances of this kind, combined with thecompounds of formula (Ia) or (Ib), selected from among fenoterol,formoterol, salmeterol,1-[3-(4-methoxybenzylamino)-4-hydroxyphenyl]-2-[4-(1benzimidazolyl)-2-methyl-2-butylamino]ethanol,1-[2H-5-hydroxy-3-oxo-4H-1,4-benzoxazin-8-yl]-2-[3-(4-N,N-dimethylaminophenyl)-2-methyl-2-propylamino]ethanol,1-[2H-5-hydroxy-3-oxo-4H-1,4-benzoxazin-8-yl]-2-[3-(4-methoxyphenyl)-2-methyl-2-propylamino]ethanol,1-[2H-5-hydroxy-3-oxo-4H-1,4-benzoxazin-8-yl]-2-[3-(4-n-butyloxyphenyl)-2-methyl-2-propylamino]ethanol,1-[2H-5-hydroxy-3-oxo-4H-1,4-benzoxazin-8-yl]-2-{4-[3-(4-methoxyphenyl)-1,2,4-triazol-3-yl]-2-methyl-2-butylamino}ethanol,optionally in the form of their racemates, their enantiomers, theirdiastereomers, as well as optionally their pharmacologically acceptableacid addition salts and hydrates. Of the betamimetics mentioned above,the compounds formoterol and salmeterol, optionally in the form of theirracemates, their enantiomers, their diastereomers, as well as optionallytheir pharmacologically acceptable acid addition salts and hydrates, areparticularly important.

The acid addition salts of the betamimetics selected from among thehydrochloride, hydrobromide, sulfate, phosphate, fumarate,methanesulfonate and xinafoate are preferred according to the invention.In the case of salmeterol, the salts selected from among thehydrochloride, sulfate and xinafoate are particularly preferred,especially the sulfates and xinafoates. In the case of formoterol, thesalts selected from among the hydrochloride, sulfate and fumarate areparticularly preferred, especially the hydrochloride and fumarate. Ofoutstanding importance is formoterol fumarate.

The corticosteroids which may optionally be used in conjunction with thecompounds of formula (Ia) or (Ib), include compounds selected from amongflunisolide, beclomethasone, triamcinolone, budesonide, fluticasone,mometasone, ciclesonide, rofleponide, GW 215864, KSR 592, ST-126,loteprednol etabonate, etiprednol dichloracetate and dexamethasone. Thepreferred corticosteroids are those selected from among flunisolide,beclomethasone, triamcinolone, loteprednol etabonate, etiprednoldichloracetate, budesonide, fluticasone, mometasone, ciclesonide anddexamethasone, while budesonide, fluticasone, loteprednol etabonate,etiprednol dichloracetate, mometasone and ciclesonide, especiallybudesonide, fluticasone, loteprednol etabonate and etiprednoldichloracetate, are of particular importance. Any reference to steroidsherein also includes a reference to salts or derivatives which may beformed from the steroids. Examples of possible salts or derivativesinclude: sodium salts, sulfobenzoates, phosphates, isonicotinates,acetates, propionates, dihydrogen phosphates, palmitates, pivalates orfuroates. The corticosteroids may optionally also be in the form oftheir hydrates.

When the corticosteroid is loteprednol etabonate, it may beadvantageously combined with an enhancing agent selected from the groupconsisting of:

-   (a) 11β,17α-dihydroxyandrost-4-en-3-one-17β-carboxylic acid    (cortienic acid, or CA);-   (b) 11β,17α-dihydroxyandrosta-1,4-dien-3-one-17β-carboxylic acid (Δ¹    cortienic acid or Δ¹-CA);-   (c) methyl 11β,17α-dihydroxyandrost-4-en-3-one-17β-carboxylate    (cortienic acid methyl ester, or MeCA);-   (d) ethyl 11β,17α-dihydroxyandrost-4-en-3-one-17β-carboxylate    (cortienic acid ethyl ester, or EtCA);-   (e) methyl 11β,17α-dihydroxyandrosta-1,4-dien-3-one-17β-carboxylate    ((Δ¹ cortienic acid methyl ester, or Δ¹-MeCA); and-   (f) ethyl 11β,17α-dihydroxyandrosta-1,4-dien-3-one-17β-carboxylate    (Δ¹ cortienic acid ethyl ester, or Δ¹-EtCA),    wherein the mole ratio of loteprednol etabonate to enhancing agent    is from about 5:1 to about 0.5:1. Such combinations with these    inactive metabolites are described in detail in WO 2005/000317 A1,    incorporated by reference herein in its entirety and relied upon.

Examples of antiallergic agents which may be used as a combination withthe compounds of formula (Ia) or (Ib) include epinastin, cetirizin,azelastin, fexofenadin, levocabastin, loratadine, mizolastin, ketotifen,emedastin, dimetinden, clemastine, bamipin, cexchloropheniramine,pheniramine, doxylamine, chlorophenoxamine, dimenhydrinate,diphenhydramine, promethazine, ebastin, desloratidine and meclizine.Preferred antiallergic agents which may be used in combination with thecompounds of formula (Ia) or (Ib) are selected from among epinastin,cetirizin, azelastin, fexofenadin, levocabastin, loratadine, ebastin,desloratidine and mizolastin, epinastin and desloratidine beingparticularly preferred. Any reference to the abovementioned antiallergicagents also includes a reference to any pharmacologically acceptableacid addition salts thereof which may exist.

When the compounds of formula (Ia) or (Ib) are used in conjunction withother active substances, the combination with steroids or betamimeticsis particularly preferred of the various categories of compoundsmentioned above.

Whether or not the compounds of formula (Ia) or (Ib) are used inconjunction with other active substances as described above, they aretypically administered in the form of a pharmaceutical compositioncomprising an anticholinergically effective amount of a compound offormula (Ia) or (Ib) and a non-toxic pharmaceutically acceptable carriertherefor. Pharmaceutically acceptable carriers, or diluents, arewell-known in the art. The carriers may be any inert material, organicor inorganic, suitable for administration, such as: water, gelatin, gumarabic, lactose, microcrystalline cellulose, starch, sodium starchglycolate, calcium hydrogen phosphate, magnesium stearate, talcum,colloidal silicon dioxide, and the like. Such compositions may alsocontain other pharmaceutically active agents, as noted above, and/orconventional additives such as stabilizers, wetting agents, emulsifiers,flavoring agents, buffers, binders, disintegrants, lubricants, glidants,antiadherents, propellants, and the like. The carrier, e.g., non-activeingredient, can be just (sterile) water with the pH adjusted to wherethe active pharmaceutical agent is very soluble. It is preferred thatthe pH be at or near 7. Alternatively and preferably, the non-activecarrier agent should be physiological saline with the pH adjustedappropriately.

The novel compounds of formula (Ia) or (Ib) can be administered in anysuitable way. The compounds can be made up in solid or liquid form, suchas tablets, capsules, powders, syrups, elixirs and the like, aerosols,sterile solutions, suspensions or emulsions, and the like.

The compounds of formula (Ia) or (Ib) can be brought into suitabledosage forms, such as compositions for administration through the oral,rectal, trandermal, parenteral, nasal, pulmonary (typically via oralinhalation) or topical (including ophthalmic) route in accordance withaccepted pharmaceutical procedures. The route of administration and thusthe dosage form will be chosen in light of the condition to be treatedwith the instant anticholinergic agents. By way of illustration only,when the compound of formula (Ia) or (Ib) is administered to treat COPDor asthma, or other serious obstructive disease of the respiratorytract, the compounds may be advantageously administered via inhalationor insufflation; for such purposes, the compounds are advantageously inthe form of an aerosol or a powder for inhalation. When administered totreat less serious respiratory disorders such as rhinitis, a nasalspray, mist or gel may be advantageous. For inducing mydriasis, anophthalmic formulation such as eye drops may be most appropriate. ForOAB, a formulation for oral administration such as tablet or capsules ora transdermal preparation may be preferred. For treating hyperhydrosis,an topical preparation formulated as an antiperspirant stick, gel,spray, cream or the like would be preferred.

For purposes of illustration, dosages are expressed based on theinhalation of an aerosol solution, such as the product AtroventInhalation Aerosol (Boehringer Ingelheim). Adjustments in dosages foradministration by other modes of inhaled administration are well knownto those skilled in the art.

In general, a therapeutically effective or anticholinergically effectiveamount of compound of formula (Ia) or (Ib) is from about 1 μg to about1,000 μg, e.g., from about 10 μg to about 1.000 μg or from about 100 μgto about 1000 μg. However, the exact dosage of the specific compound offormula (Ia) or (Ib) will vary depending on its potency, the mode ofadministration, the age and weight of the subject and the severity ofthe condition to be treated. The daily dosage may, for example, rangefrom about 0.01 μg to about 10 μg per kg of body weight, administeredsingly or multiply in doses e.g. from about 1 μg to about 1,000 μg each.The compounds of formula (Ia) or (Ib) can be administered from one tofour times daily, e.g., once or twice daily.

The dosage form for inhalation can be an aerosol. The minimum amount ofan aerosol delivery is about 0.2 ml and the maximum aerosol delivery isabout 5 ml. The concentration of the compounds of formula (Ia) or (Ib)may vary as long as the total amount of spray delivered is within theabout 0.2 to about 5 ml amount and as long as it delivers ananticholinergically effective amount of the compound of formula (Ia) or(Ib). It is well known to those skilled in the art that if theconcentration is higher, one gives a smaller dose to deliver the sameeffective amount.

The dosage form for inhalation can also be via intranasal spray. Theminimum amount of an aerosol delivery is about 0.02 ml per nostril andthe maximum aerosol delivery is about 0.2 ml per nostril. Theconcentration of the compounds of formula (Ia) or (Ib) may vary as longas the total amount of spray delivered is within about 0.02 ml pernostril to about 0.2 ml per nostril, e.g., between about 0.05 ml pernostril and about 0.08 ml per nostril, and it delivers ananticholinergically effective amount of the compound of formula (Ia) or(Ib).

Of course, the volume of aerosol or intranasal spray for delivering ananticholinergically effective amount of the compound of formula (Ia) or(Ib) depends upon the concentration of the compound in the aerosol orintranasal spray, i.e., higher concentrations of the compound of formula(Ia) or (Ib) require smaller dosage volumes to deliver a therapeuticallyeffective amount and lower concentrations of the compound of formula(Ia) or (Ib) require larger dosage volumes to deliver the sameanticholinergically effective amount.

Aerosols for inhalation of various pharmaceutical agents are well knownto those skilled in the art, including many aerosols for treatingasthma. Aerosols may be produced with a nebulizer. Typically, thenebulizer is charged with a carrier solution and the compound of formula(Ia) or (Ib) in an amount sufficient to effectively deliver ananticholinergically effective amount of the compound of formula (Ia) or(Ib). For instance, depending upon the nebulizer and its operatingconditions, the nebulizer may be charged with several hundred mg ofanticholinergic compound in order to deliver about 1 μg to about 1000μg, e.g., from about 10 μg to about 1000 μg or from about 50 μg to about500 μg, of the compound of formula (Ia) or (Ib).

The dosage form for inhalation may also be in powder form. Powders forinhalation of various pharmaceutical agents are well known to thoseskilled in the art, including many powders for treating asthma. When thedosage form is a powder, the compounds of formula (Ia) or (Ib) can beadministered in pure form or diluted with an inert carrier. When aninert carrier is used, the compounds are compounded such that the totalamount of powder delivered delivers an “effective amount” of thecompounds according to the invention. The actual concentration of theactive compound may vary. If the concentration is lower, then morepowder must be delivered, if the concentration is higher, less totalmaterial must be delivered to provide an effective amount of the activecompound according to the invention. Any of the foregoing pharmaceuticalcompositions may further comprise one or more additional activesubstances, particularly corticosteroids and/or betamimetics asdiscussed earlier.

“Pharmaceutically acceptable” refers to those properties and/orsubstances which are acceptable to the patient from apharmacological/toxicological point of view and to the manufacturingpharmaceutical chemist from a physical/chemical point of view regardingcomposition, formulation, stability, patient acceptance andbioavailability.

Suitable preparations for administering the compounds of formula (Ia) or(Ib) include tablets, capsules, suppositories, solutions, etc. Ofparticular importance (particularly when treating asthma or COPD orother respiratory disorders) is the administration of the compounds byinhalation. The proportion of pharmaceutically active compound orcompounds should be in the range from 0.05 to 90% by weight, preferably0.1 to 50% by weight of the total composition. Suitable tablets may beobtained, for example, by mixing the active substance(s) with knownexcipients, for example inert diluents such as calcium carbonate,calcium phosphate or lactose, disintegrants such as corn starch oralginic acid, binders such as starch or gelatin, lubricants such asmagnesium stearate or talc and/or agents for delaying release, such ascarboxymethyl cellulose, cellulose acetate phthalate, or polyvinylacetate. The tablets may also comprise several layers. Tablets and othersolid oral formulations are of particular interest in the treatment ofOAB or ulcers while opthalmic solutions, suspensions and gels are ofspecial interest for inducing mydriasis and topical gels, solids andsprays are of particular use as antiperspirants.

Coated tablets may be prepared accordingly by coating cores producedanalogously to the tablets with substances normally used for tabletcoatings, for example collidone or shellac, gum arabic, talc, titaniumdioxide or sugar. To achieve delayed release or preventincompatibilities the core may also consist of a number of layers.Similarly the tablet coating may consist of a number or layers toachieve delayed release, possibly using the excipients mentioned abovefor the tablets.

Syrups or elixirs containing the active substances of formulas (Ia) or(Ib) or combinations thereof as described above may additionally containa sweetener such as saccharin, cyclamate, aspartame, sucralose, glycerolor sugar and a flavor enhancer, e.g. a flavoring such as vanillin ororange extract. They may also contain suspension adjuvants or thickenerssuch as sodium carboxymethyl cellulose, wetting agents such as, forexample, condensation products of fatty alcohols with ethylene oxide, orpreservatives such as p-hydroxybenzoates.

Solutions are prepared in the usual way, e.g. with the addition ofisotonic agents, preservatives such as p-hydroxybenzoates, orstabilizers such as alkali metal salts of ethylenediamine tetraaceticacid, optionally using emulsifiers and/or dispersants, while if water isused as the diluent, for example, organic solvents may optionally beused as solvating agents or dissolving aids, and transferred intoinjection vials or ampules or infusion bottles.

Capsules containing one or more active substances or combinations ofactive substances may for example be prepared by mixing the activesubstances with inert carriers such as lactose or sorbitol and packingthem into gelatin capsules. Suitable suppositories may be made forexample by mixing with carriers provided for this purpose, such asneutral fats or polyethyleneglycol or the derivatives thereof.Excipients which may be used include, for example, water,pharmaceutically acceptable organic solvents such as paraffins (e.g.petroleum fractions), vegetable oils (e.g. groundnut or sesame oil),mono- or polyfunctional alcohols (e.g. ethanol or glycerol), carrierssuch as e.g. natural mineral powders (e.g. kaolins, clays, talc, chalk),synthetic mineral powders (e.g. highly dispersed silicic acid andsilicates), sugars (e.g. cane sugar, lactose and glucose), emulsifiers(e.g. lignin, spent sulfite liquors, methylcellulose, starch andpolyvinylpyrrolidone) and lubricants (e.g. magnesium stearate, talc,stearic acid and sodium lauryl sulphate).

The preparations are administered by the usual methods, preferably byinhalation in the treatment of asthma or COPD or other respiratorydisorders. For oral administration the tablets may, of course, contain,apart from the above-mentioned carriers, additives such as sodiumcitrate, calcium carbonate and dicalcium phosphate together with variousadditives such as starch, preferably potato starch, gelatin and thelike. Moreover, lubricants such as magnesium stearate, sodium laurylsulfate and talc may be used at the same time for the tablettingprocess. In the case of aqueous suspensions the active substances may becombined with various flavor enhancers or colorings in addition to theexcipients mentioned above.

The dosage of the compounds of formula (Ia) and (Ib) is naturallygreatly dependent on the route of administration and the complaint to betreated. When administered by inhalation the compounds of formula (Ia)or (Ib) are characterized by high efficacy even at doses in the μgrange. The compounds of formula (Ia) or (Ib) can also be usedeffectively above the μg range. The dosage may then be in the gramrange, for example. Particularly when administered by a method otherthan inhalation, the compounds according to the invention may be givenin higher doses (in the range from 1 to 1000 mg, for example, althoughthis does not imply any limitation).

The compounds of formula (Ia) and (Ib), combinations of a compound offormula (Ia) or (Ib) with one or more other active agents, andcompositions comprising a compound of formula (Ia) or (Ib), with orwithout one or more other active agents, as described hereinabove arethus useful in a method for eliciting an anticholinergic response in asubject in need of same, comprising administering to said subject ananticholinergically effective amount of said compound or composition. Inparticular embodiments, the method is for treating an obstructivedisease of the respiratory tract, especially when the disease is chronicobstructive pulmonary disease or asthma, or for treating overactivebladder. In another embodiment, the method comprises inducing mydriasisin the eye(s) of a subject in need of such treatment, comprisingtopically applying to the eye(s) of said subject a mydriaticallyeffective amount of a compound of formula (Ia) or (Ib) or combination orcomposition comprising it as described hereinabove. Use of compounds offormula (Ia) or (Ib) in the preparation of a medicament for treating acondition responsive to an anticholinergic agent (such as any of theseconditions disclosed above) is likewise provided herein.

In particular embodiments there are provided combinations of thecompound of formula (Ia) or (Ib) with other active agents, especiallyone or more antiinflammatory corticosteroids, betamimetic agents orantiallergic agents. In the combination products, the active agents arepresent in a combined amount effective to treat the target condition,especially to treat an obstructive disease of the respiratory tract,most especially to treat chronic obstructive pulmonary disease orasthma. In preferred embodiments, the other active agent is abetamimetic agent or an antiinflammatory corticosteroid. Of particularinterest are combinations of a compound of formula (Ia) or (Ib) and acorticosteroid, especially loteprednol etabonate or etiprednoldichloracetate. When loteprednol etabonate is selected as thecorticosteroid, its activity can be enhanced by combination withcortienic acid or Δ¹-cortienic acid or a methyl or ethyl ester ofcortienic acid or Δ¹-cortienic acid, in a mole ratio of from about 5:1to about 0.5:1. A molar ratio of about 1:1, which can be approximated bya 1:1 ratio by weight, is particularly convenient.

Initial Studies

Materials and Methods

Materials

Glycopyrrolate (glycopyrronium bromide) was kindly provided byBoehringer Ingelheim Chemicals, Inc. Carbamylcholine bromide(carbachol), atropine methylbromide (atropine MeBr), and scopolaminemethylbromide (scopolamine MeBr) were obtained from Sigma Chemicals Co.(St. Louis, Mo.). N-[³H]-Methyl-scopolamine (NMS) was obtained fromAmersham Biosciences UK Limited (Buckinghamshire, UK). Cloned humanmuscarinic receptor subtypes M₁-M₄ were obtained from Applied CellScience Inc. (Rockville, Md.). Scintiverse BD was from Fisher ScientificCo. (Pittsburgh, Pa.).

Chemicals used for synthesis were reagent or HPLC grade, and wereobtained from Aldrich (Milwaukee, Wis.) and Fisher Scientific Co.Melting points were taken on Fisher-Johns melting apparatus. NMR spectrawere recorded on a Bruker Advance 500 MHz NMR spectrometer and arereported in ppm relative to TMS. Elemental analyses were performed byAtlantic Microlab Inc (Atlanta, Ga.).

Synthesis Racemic Cyclopentylmandelic Acid (1)

Cyclopentylmagnesium bromide ether solution (100 ml, 2M; 0.2 mol) wasadded drop-wise to benzoylformic acid (15 g, 0.1 mol) in 330 ml ofanhydrous ethyl ether at 0° C. The mixture was stirred at 0° C. for 30min and at room temperature for 24 h. The reaction mixture was treatedwith 1 N HCl, and the aqueous solution was extracted with ether. Thecombined ether solution was treated with K₂CO₃ solution. The potassiumcarbonate solution was acidified with HCl and extracted with ethertwice. The ether solution was dried with anhydrous sodium sulfate andevaporated to give a crude product. The crude product was washed withwater to get pure racemic cyclopentylmandelic acid 1 (8.0 g, 36.4%).Needle-like crystals, m.p.: 153-154° C. ¹H NMR (CDCl₃, 500 MHz):1.28-1.39, 1.42-1.50, 1.51-1.61, 1.63-1.72 [8H, m, (CH₂)₄], 2.93 [1H, p,CHC(OH)], 7.26-7.30, 7.33-7.36, 7.65-7.67 (5H, m, Ph) ppm.

Methyl cyclopentylmandelate (2)

To a mixture of racemic cyclopentylmandelic acid R/S(±)1 (4.47 g, 20mmol) and potassium carbonate (7.01 g, 50 mmol) in DMF (50 ml), methyliodide (8.64 g, 60 mmol) was added at room temperature. The mixture wasstirred at room temperature for 2 h, and then poured into water andextracted with hexanes three times. Evaporation of the dried hexanesextract gave a crude product. Flash chromatography of the crude producton silica gel with 1.5:1 hexanes:methylene chloride gave the pureproduct 2 (3.02 g, 64%). ¹H NMR (CDCl₃, 300 MHz): 1.32-1.37, 1.43-1.69[8H, m, (CH₂)₄], 2.90 [1H, p, CHC(OH)], 3.74 (1H, s, OH), 3.77 (3H, s,CH₃), 7.25-7.37, 7.63-7.65 (5H, m, Ph) ppm.

N-Methyl-3-pyrrolidinyl cyclopentylmandelate (4)

A solution of 2 (2.20 g, 9.4 mmol) and N-methyl-3-pyrrolidinol (0.3,1.30 g, 13 mmol) in 40 ml of n-heptane was heated until 20 ml of heptanehad been distilled. About 0.003 g of sodium was added, and the solutionwas stirred and heated for 2 h as the distillation was continued. Moreheptane was added at such a rate as to keep the reaction volumeconstant. Additional sodium was added at the end of an hour. Thesolution was then cooled and extracted with 3N HCl. The acid extract wasmade alkaline with concentrated NaOH and extracted three times withether. Removal of the dried ether solution gave a crude oil. Flashchromatography of the crude product on silica gel with 8:1 EtOAc:EtOHgave pure product 4 (2.053 g, 72%). Analysis for C₁₈H₂₅NO₃. Calcd: C,71.26; H, 8.31; N, 4.62. Found: C, 71.55; H, 8.44; N, 4.68. ¹H NMR(CDCl₃, 500 MHz): 1.27-1.35, 1.40-1.47, 1.54-1.60, 1.75-1.90 [8H, m,(CH₂)₄], 2.12-2.30, 2.52-2.57, 2.64-2.81 (6H, m CH₂NCH₂CH₂), 2.33, 2.36(3H, 2s, NCH₃), 2.93 [(1H, p, CHC(OH)], 3.83 (1H, bs, OH), 5.23 (1H, m,CO₂CH), 7.23-7.36, 7.64-7.67 (5H, m, Ph) ppm.

3-(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide, Compound (a)

To compound 4 (0.8235 g, 2.71 mmol) in 30 ml of dry acetonitrile, methylbromoacetate (1.08 g, 7.06 mmol) was added at room temperature. Themixture was stirred for 2 h. Evaporation of acetonitrile gave a crudeproduct. The crude product was dissolved in a small volume of methylenechloride and then poured into 100 ml of dry ethyl ether to precipitate.This procedure was repeated three times to obtain Compound (a) as pureproduct (0.9912 g, 80%). White powder, m.p.: 192-194° C. Analysis forC₂₁H₃₀BrNOs. Calcd: C, 55.27; H, 6.63; N, 3.07. Found: C, 55.11; H,6.59; N, 3.03. ¹H NMR (CDCl₃, 500 MHz): 1.23-1.29, 1.31-1.37, 1.41-1.47,1.53-1.67 [8H, m, (CH₂)], 2.18-2.23, 2.73-2.80, 4.04-4.16, 4.21-4.25(6H, m, CH₂NCH₂CH₂), 2.85 [1H, p, CHC(OH)], 3.57 (3H, s, NCH₃), 3.80(3H, s, CO₂CH), 4.66, 4.85 (2H, 2dd, CH₂CO₂), 5.27 (1H, s, OH), 5.52(1H, m, CO₂CH), 7.25-728, 7.32-7.35, 7.57-7.59 (5H, m, Ph) ppm.

3-(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide, Compound (b)

To compound 4 (0.369 g, 122 mmol) in 10 ml of dry acetonitrile, ethylbromoacetate (0.377 g, 2.25 mmol) was added at room temperature. Themixture was stirred for 2 h. Evaporation of acetonitrile gave a crudeproduct. The crude product was dissolved in a small volume of ethylenechloride and then poured into 50 ml of dry ethyl ether to precipitate.This procedure was repeated three times to obtain Compound (b) as pureproduct (0.45 g, 79%). White powder, m.p.: 192-194° C.

Analysis for C₂₂H₃₂BrNO₅. Calcd: C, 56.17; H, 6.86; N, 2.98. Found: C,56.14; H, 6.89; N, 2.94. ¹H NMR (CDCl₃. 500 MHz): 1.35 (3H, t, CH₃CH₂),1.26-1.33, 1.42-1.47, 1.55-1.67 [8H, m, (CH₂)₄], 2.14-2.21, 2.73-2.79,4.12-4.17, 4.22-4.29 (6H, m, CH₂NCH₂CH₂), 2.86 [1H, p, CHC(OH)], 3.62(3H, s, NCH₃), 4.25 (2H, q, CH₃CH₂), 4.67, 4.83 (2H, dd, CH₂CO₂), 4.91(1H, s, OH), 5.53 (1H, m, CO₂CH), 7.25-7.27, 7.32-7.34, 7.57-7.59 (5H,m, Ph) ppm.

Resolution of racemic cyclopentylmandelic acid (1)

(−)-Strychnine (6.10 g) in 50 ml of methanol (suspension) was added toracemic cyclopentylmandelic acid 1. (3.96 g) in methanol (20 ml) at roomtemperature. The reaction solution was let to stand for overnight. Thecrystals were removed by filtration and crystallized again with hotmethanol. The second crop of crystals was collected by filtration andtreated with sodium hydroxide solution. The basic solution was extractedwith methylene chloride twice (methylene chloride solution discarded),and then acidified with hydrochloric acid to recover the resolvedcyclopentylmandelic acid. To this resolved acid (20.6 mg in 0.1 ml ofethyl acetate), 13 μL of (+)-α-phenylethylamine was added. Theprecipitate which formed was washed with hexane three times and driedunder vacuum. The precipitate was identified by NMR as optically purecyclopentylmandelic acid, R(−)1, (1.49 g, 37.6%). M.p.: 121-122° C.[α]^(25°) _(D)=−22.5° (c=1 g/100 ml, CHCl₃). ¹H NMR (CDCl₃, 500 MHz):1.28-1.39, 1.42-1.50, 1.51-1.61, 1.64-1.73 [8H, m, (CH₂)₄], 2.93 [1H, p,CHC(OH)], 7.25-7.28, 7.32-7.35, 7.64-7.65 (5H, m, Ph) ppm.

Methyl (−)-cyclopenylmandelate, R(−)₂

To a mixture of (−)-cyclopentylmandelic acid, R(−)1, (1.83 g, 8.3 mmol)and potassium carbonate (2.87 g, 21 mmol) in DMF (21 ml), methyl iodide(3.53 g, 25 mmol) was added at room temperature. The mixture was stirredat room temperature for 2 h, and then poured into water and extractedwith hexanes three times. Evaporation of the dried hexanes extract gavea crude product. Flash chromatography of the crude product on silica gelwith 1.5:1 hexanes:methylene chloride gave pure product R(−)₂ (1.95 g,100%). Analysis for C₁₈H₁₈O₃. Calcd: C, 71.77; H, 7.74. Found: C, 71.88;H, 7.80. ¹H NMR (CDCl₃, 500 MHz): 1.32-1.36, 1.43-1.61 [8H, m, (CH₂)₄],2.90 [1H, p, CHC(OH)], 3.71 (1H, s, OH), 3.79 (3H, s, CH₃), 7.25-7.28,7.31-7.35, 7.63-7.65 (5H, m, Ph) ppm.

N-Methyl-3-pyrrolidinyl (−)-cyclopentylmandelate, 2R-4

A solution of R(−)₂ (1.85 g, 7.9 mmol) and N-methyl-3-pyrrolidinol (3,1.05 g, 10.4 mmol) in 40 ml of n-heptane was heated until 20 ml ofheptane had distilled. Approximately 0.003 g of sodium was added, andthe solution was stirred and heated for 2 h as the distillation wascontinued. More heptane was added at such a rate as to keep the reactionvolume constant. Additional sodium was added at the end of an hour. Thesolution was then cooled and extracted with 3N HCl. The acid extract wasmade alkaline with concentrated NaOH and extracted three times withether. Removal of dried ether solution gave a crude oil. Flashchromatography of the crude product on silica gel with 8:1 EtOAc:EtOHgave 2R-4 as a mixture of two diastereoisomers in an NMR-estimated ratioof 1:1, (1.68 g, 70%). Analysis for C₁₈H₂₅NO₃.0.2H₂O. Calcd: C, 70.42;H, 8.34; N, 4.5. Found: C, 70.60; H, 8.26; N, 4.63. ¹H NMR (CDCl₃, 500MHz): 1.28-1.37, 1.40-1.47, 1.51-1.70, 1.73-1.80, 1.83-1.90 [8H, m,(CH₂)₄], 2.14-2.21, 2.27-2.35, 2.36-2.42, 2.52-2.55, 2.64-2.81 (6H, m,CH₂NCH₂CH₂), 2.33, 2.37 (3H, 2s, NCH₃), 2.93 [1H, p, CHC(OH)], 3.78 (1H,bs, OH), 5.22 (1H, m CO₂CH), 7.24-7.27, 7.31-7.35, 7.64-7.66 (5H, m, Ph)ppm.

(2R)3-(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide, Compound (c)

To compound 2R-4 (0.15 g, 0.49 mmol) in 6 ml of dry acetonitrile, methylbromoacetate (0.194 g, 1.27 mmol) was added at room temperature. Themixture was stirred for 6 h. Evaporation of acetonitrile gave a crudeproduct. The crude product was dissolved in a small volume of methylenechloride and then poured into 50 ml of dry ethyl ether to precipitate.This procedure was repeated three times to obtain the product, Compound(c) (0.1879 g, 83%), as a mixture of four diastereoisomers in anNMR-estimated ratio of 1:1:2:2. White powder, m.p.: 153-155° C.[α]^(25°) _(D)=+0.5° (c=1 g/100 ml CHCl₃). Analysis forC₂₁H₃₀BrNO₅.0.2H₂O. Calcd: C, 54.86; H, 6.62; N, 3.05. Found: C, 54.75;H, 6.66; N, 3.01. ¹H NMR (CDCl₃, 500 MHz): 130-1.37, 1.41-1.50,1.55-1.73 [8H, m, (CH₂)₄], 1.93-2.00, 2.12-2.26, 2.75-2.95, 3.00-3.03,4.30-4.50, 4.57-4.61 [7H, m, CHC(OH) and CH₂NCH₂CH₂], 3.09, 3.30 (1H,2s, OH), 3.64, 3.66, 3.84, 3.95, 3.97 (3H, is, NCH₃), 3.74, 3.77, 3.79,3.81 (3H, 4s, CO₂CH₃), 4.78, 4.83; 4.90, 4.97; 5.30, 5.35; 5.37, 5.41(2H, 4 groups of 2dd, CH₂CO₂), 5.53 (1H, m, CO₂CH), 7.23-7.29,7.31-7.38, 7.56-7.60 (5H, m, Ph) ppm.

(2R)3-(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide, Compound (d)

To compound 2R-4 (0.22 g, 0.73 mmol) in 10 ml of dry acetonitrile, ethylbromoacetate (0.21 ml, 0.316 g, 1.89 mmol) was added at roomtemperature. The mixture was stirred for 22 hours. Removal ofacetonitrile gave a crude product. The crude product was dissolved insmall volume of ethylene chloride and then poured into 50 ml of dryethyl ether to precipitate. This procedure repeated three times toobtain the product, Compound (d) (0.3085 g, 90%) as a mixture of fourdiastereoisomers in an NMR-estimated ratio of 1:1:2:2. White powder,m.p.:143-145° C. [α]^(25°) _(D)=+5.6° (c=1 g/100 ml CHCl₃). Analysis forC₂₂H₃₂BrNO₅.0.3H₂O. Calcd: C, 55.53; H, 6.91; N, 2.94. Found: C, 55.46;H, 6.85; N, 2.97. ¹H NMR (CDCl₃, 500 MHz): 1.26, 1.28, 1.32, 1.35 (3H,4t, CH₃CH₂), 1.44-1.50, 1.53-1.63, 1.65-1.70 [8H, m, (CH₂)₄], 1.93-2.00,2.04-2.11, 2.18-2.25, 2.76-2.96, 3.01-3.04, 4.09-4.26 [7H, m, CHC(OH)and CH₂NCH₂CH₂], 3.06, 3.28 (1H, 2s, 01-1), 3.66, 3.69, 3.81, 3.82,3.94, 3.96 (3H, 6s, NCH₃), 4.61, 4.69; 4.76, 4.85; 5.17, 5.22; 5.26,5.30 (2H, 4 set of dd, CH₂CO₂), 4.26-4.52 (2H, m, CH₃CH₂), 5.53 (1H, m,CO₂CH), 7.24-7.29, 7.31-7.38, 7.56-7.60 (5H, m, Ph) ppm.

pH Profile

The stabilities of the soft glycopyrrolates in standard phosphatebuffers (0.05 M) of various pH (pH 6.00-8.40) were investigated at 37′C.Aliquots of 4.4 mM of the compounds in water solution were added to thebuffer solutions to give a final concentration of 0.44 mM. Atappropriate time intervals, samples were taken and analyzed by HPLC tomonitor the disappearance of the soft analogs and the formation of itshydrolysis products. The pseudo-first-order rate constant (k, min⁻¹) andhalf-life (t_(1/2), min) of the disappearance of the compound in thebuffer were calculated.

In Vitro Studies

The stability of soft glycopyrrolates in biological media in vitro wasdetermined by measuring the pseudo-first-order rate constants (k, min⁻¹)and half-lives (t_(1/2), min) of the disappearance of the compound inrat blood and plasma. Aliquots of 22 mM were added to the biologicalmedium at 37° C. to yield a final concentration of 0.7 mM. Atappropriate time intervals, samples (0.15 ml) were withdrawn and mixedwith 0.3 ml of 5% dimethylsulfoxide in acetonitrile solution. Themixtures were centrifuged, and the supernatants were analyzed by HPLC.Experiments were performed in triplicates.

Analytical Method

The HPLC system used for the analysis of the compounds of formula (I)and their hydrolysis products was as follows: A Supelcosil LC-8 column(25 cm×4.6 mm) was used with a mobile phase of acetonitrile (42%) andaqueous solution (58%) containing sodium phosphate (10 mM), acetic acid(0.1%), and triethylamine (0.1%). At a flow rate of 1 ml/min, theretention times were 6.02 min for Compounds (a) and (c), 7.27 min forCompounds (b) and (d) and 4.14 min (hydrolysis product), respectively.With an injection volume of 10 μl, the detection limit was 1 μg/ml.

Receptor Binding Affinity

Receptor binding studies were performed with N-[³H]-methylscopolamine(NMS) in assay buffer (phosphate-buffered saline, PBS, without Ca⁺⁺ orMg⁺⁺, pH 7.4) following the protocol obtained from Applied Cell ScienceInc. (Rockville, Md.). A 10 mM NaF solution was added to the buffer asan esterase inhibitor. The assay mixture (0.2 ml) contained 20 μldiluted membranes (receptor proteins, final concentration: M₁, 38 μg/ml;M₂, 55 μg/ml; M₃, 27 μg/ml; and M₄, 84 μg/ml). The final concentrationof NMS for the binding studies was 0.5 nM. Specific binding was definedas the difference in [³H]NMS binding in the absence and presence of 5 μMatropine for M₁ and M₂ or 1 μM atrophic for M₃ and M₄. Incubation wascarried out at room temperature for 120 min. The assay was terminated byfiltration through a Whatman GF/C filter (presoaked with 0.5%polyethyleneimine). The filter was then washed six times with 1 ml icecold buffer (50 mM Tris-HCl, pH 7.8, 0.9% NaCl), transferred to vials,and 5 ml of Scintiverse was added. Final detection was performed on aPackard 31800 liquid scintillation analyzer (Packard Instrument Inc.,Downer Grove, Ill.). Data obtained from the binding experiments werefitted to the %[³H] NMS bound 100−[100x^(n)/k/(1+x^(n)/k)] equation, toobtain the Hill coefficient n, and then to %[³H] NMSbound=100−[100x^(n)/IC₅₀/(1+x^(n)/IC₅₀)], to obtain IC₅₀s (x being theconcentration of the tested compound). Based on the method of Cheng andPrusoff (Cheng & Prusoff 1973), K_(i) was derived from the equationK_(i)═IC₅₀/(1+L/K_(d)), where L is the concentration of the radioligand.IC₅₀ represents the concentration of the drug causing 50% inhibition ofspecific radioligand binding, and K_(d) represents the dissociationconstant of the radioligand receptor complex. Experiments were performedin triplicates. Data were analyzed by a non-linear least-squarecurve-fitting procedure using Scientist software (MicroMath Inc., SaltLake City, Utah).

pA₂ Values

Male guinea pigs obtained from Harlan Sprague Dawley Inc. (Indianapolis,Ind.) and weighing about 400 g were used after overnight fasting.Animals were sacrificed by decapitation, and the ileum (the region of 5cm upward of the cecum) was isolated and removed. The ileum was cut into2.5 cm pieces and suspended in an organ bath containing 30 ml of mixtureof Tyrode's solution and 0.1 mM hexamethonium bromide. The organ bathwas constantly aerated with oxygen and kept at 37° C. One end of theileum strip was attached to a fixed support at the bottom of the organbath, and the other end to an isometric force transducer (Model TRN001,Kent Scientific Corp., Conn.) operated at 2-10 g range. The ileum stripwas kept at a 2 g tension, and carbachol was used as antagonist. Theileum contracted cumulatively upon the addition of consecutive doses ofcarbachol (10-20 μl of 2×10⁻⁴−2×10⁻³ M in water solution). Contractionswere recorded on a physiograph (Kipp & Zonen Flarbed Recorder, Holland).After the maximum response was achieved, the ileum was washed threetimes, and a fresh Tyrode's solution containing appropriateconcentration of the antagonist [Compound (a), (b), (c) or (d),glycopyrrolate, or scopolamine] was replaced. An equilibration time of10 min was allowed for the antagonists before the addition of carbachol.Four to six trials were performed for each antagonist.

Pharmacological Activities of Soft Glycopyrrolates

The mydriatic effects of the soft drugs (a), (b), (c) and (d) in rabbiteyes have been compared with that of glycopyrrolate. Four healthy maleNew-Zealand white rabbits weighting about 3.5 kg were used. Toinvestigate the dose-mydriatic-response relationships, 100 μl of variousconcentrations of the compounds (0, 0.5, and 1% for the soft drugs and0, 0.05, 0.1, and 0.2% for glycopyrrolate) were administered in the eyesto determine the pharmacodynamically equivalent doses, the lowest dosesthat induce the maximum pupil dilations. Drug solutions were applied toone eye; only water was applied to the other eye that served as control.Experiments were carried out in a light- and temperature-controlledroom. At appropriate time intervals, the pupil diameters of both eyeswere recorded. Difference in pupil diameters between each time-point andzero time-point were calculated for both treated and control eyes andreported as mydriatic responses [(treated-control)/control in %].Control eye dilations were monitored to determine whether systemicabsorption had occurred or not. For each compound, four trials have beenconducted. Animal studies were performed in accordance with the Guidefor the Care and Use of Laboratory Animals adopted by the NationalInstitute of Health, USA. Institutional animal care and use committee(IACUC) approval was obtained prior to the initiation of this researchand during its execution.

Statistical Analysis

Stability, receptor binding, and pA₂ activities were compared using botht-tests and nonparametric Mann-Whitney U tests for the compound-pairs ofinterest. Pharmacological activities (maximum response R_(max) % andarea under the effect curves AUC^(eff)) were compared using ANOVAfollowed by Tukey-Kramer multiple comparison tests as a parametric posthoc test (Jones 2002), A significance level of p<0.05 was used in allcases. All statistical analyses were performed using NCSS (NumberCruncher Statistical Systems, Kaysville, Utah, USA).

Results and Discussion

Synthesis

The new soft glycopyrrolate derivatives, compounds (a) and (b)[3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(alkoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; alkoxy being methoxy and ethoxy for (a) and (b), respectively],have been synthesized as shown in Scheme 1 except for the second,resolution step. This involved (i) Grignard reaction ofcyclopentylmagnesium bromide with benzoylformic acid in anhydrous etherto give the racemic cyclopentylmandelic acid 1; (ii) methylation of 1with methyl iodide and potassium carbonate in DMF at room temperature toyield methyl cyclopentylmandelate 2; (iii) transesterification of 2 with1-methyl-3-pyrrolidinol 3 in heptane to give N-methyl-3-pyrrolidinylcyclopentylmandelate 4; and (iv) quaternization of 4 with alkylbromoacetate in acetonitrile to give the final product 5 [Compound (a)or Compound (b)]. These are racemic soft glycopyrrolate derivatives, andthey have been characterized by NMR and elemental analysis.

Because stereospecificity is known to be important at muscarinicreceptors, improved anticholinergic activity being obtained with the 2Rconfiguration of glycopyrrolate-type substances, these soft drugcandidates have also been prepared starting with optically purecyclopentylmandelic acid, R(−)1. Racemic 1 was resolved by repeatedcrystallization of the salts produced between acid 1 and (−)-strychnine.Optically pure free acid was recovered by basification of the salts withsodium hydroxide solution followed by acidification with hydrochloricacid. The obtained left rotatory)(−22.5° optically pure R(−)1 wascharacterized by NMR. Grover and coworkers reported the highlystereoselective synthesis of S)-cyclopentyl-phenylglycoxilic acid using(S)-mandelic acid in 2000, and they found(S)-cyclopentyl-phenylglycoxilic acid to have positive optical rotation,Accordingly, R(−)1, which was found to have an optical rotation of[α]=−22.5°, is the R form. The NMR of the salt formed by the resolvedcyclopentylmandelic acid R(−)1 and (+)-α-phenylethylamine gave a singlepentaplet for the CHC(OH) group; whereas the salt of the unresolved 1and (+)-α-phenylethylamine gave two pentaplets for CHC(OH).

The soft glycopyrrolate Compounds (c) and (d) having 2R configurationshave been synthesized from R(−)cyclopentylmandelic acid R(−)1 by theroute shown in Scheme 1, and they were also characterized by NMR andelemental analysis. The optical rotations of Compound (c) and Compound(d) were +0.5° and +5.6°, respectively.

The racemic Compound (a) and Compound (b) had much simpler NMR spectrathan the corresponding resolved compounds, Compound (c) and Compound(d). These molecules have a total of three chiral centers as shown inScheme 1. In Compound (c) and Compound (d), one of the chiral centerswas resolved, but two others remained; hence, they both are mixture offour diastereoisomers complicating their NMR spectra. For example, theCH₃CH₂ methyl group showed only one triplet at 1.35 ppm in Compound (b),where it is not subject to unequal chemical environments; however, itshowed four triplets at 1.26, 1.28, 1.32, and 1.35 ppm, respectively inCompound (d), which has one resolved and two unresolved chiral centersand is a mixture of four diastereoisomers (RPR, RSR, RRS, RSS). Also,the AB system of Compound (b)'s CH₂CO₂ group showed one set ofdouble-doublet signals at 4.67 and 4.83 ppm, but the same system inCompound (d) showed four sets of double-doublet signals at 4.61, 4.69;4.76, 4.85; 5.17, 5.22; and 5.26, 5.30 ppm.

pH Profile

In the pH range of 6.00-8.40 and at 37° C., the chemical hydrolysis ofthe present soft glycopyrrolate compounds was significantlypH-dependent. As shown in FIG. 1, they are more stable under acidiccondition, and the ethyl derivatives are more stable than thecorresponding methyl derivatives. The half-lives of Compounds (a), (c),(b) and (d) in aqueous solutions at pH 6.0 were 91, 77, 155, and 134 h,respectively. However, at pH 8.4, the corresponding half-lives decreasedto 8, 7, 16, and 12 min, respectively. The pH profiles are displayed inFIG. 1, and the results indicate a base-catalyzed hydrolysis of thecompounds with a correlation coefficient of 0997-0.998. For illustrativepurposes, the time-profile of the disappearance of Compound (c) and theconcurrent formation of the corresponding acid at pH 7.4 is shown inFIG. 2.

In Vitro Stability

In vitro stability studies have been performed using rat blood andplasma by measuring the pseudo-first-order rate constant (k, min⁻¹) andhalf-life (t_(1/2), min) of the disappearance of the parent compounds(Table 1). At 37′C and pH 7.4, the hydrolysis of soil glycopyrrolateanalogs was relatively fast in plasma with half-lives of 19.5, 20, 44,and 34 min for Compounds (a), (c), (b) and (d), respectively, andsignificantly slower in blood (57, 57, 97, and 86 min, respectively;p<0.05 for all compounds, t-test or nonparametric Mann-Whitney U test),indicating that blood cell binding is significant enough to slow thehydrolytic degradation of these esters. The ethyl esters were morestable than the methyl derivatives (p<0.05, t-test or nonparametricMann-Whitney U test).

TABLE 1 Pseudo-first-order rate constant (k, min⁻¹) and half-life(t_(1/2), min) for the disappearance of soft analogs in rat plasma andblood. Data represent mean ± SD of three experiments. Compound Medium k× 10⁻³, min⁻¹ t_(1/2), min r² (a) plasma 36.4 ± 5.0 19.5 ± 2.7 0.998blood 12.3 ± 1.3 57.0 ± 5.8 0.998 (b) plasma 15.8 ± 0.2 44.0 ± 0.4 0.997blood  7.2 ± 0.2 96.6 ± 2.9 0.996 (c) plasma 34.5 ± 3.2 20.0 ± 2.1 0.993blood 12.4 ± 1.4 56.7 ± 6.1 0.997 (d) plasma 20.9 ± 3.0 33.8 ± 4.8 0.998blood  8.0 ± 0.1 86.4 ± 1.2 0.998In vitro Pharmacodynamic Evaluation

To evaluate the relative potency of the newly synthesized soft analogs,receptor binding affinities, pK_(i), and guinea pig ileum contractionability, pA₂, were determined.

Receptor Binding Studies

The receptor binding affinities of the compounds determined byradioligand binding assays using human cloned muscarinic receptorsubtypes, M₁-M₄, are presented in Table 2. The 2R isomers, Compounds (c)and (d), had pK_(i) values that are in the 8.7-9.5 range; somewhat less,but close to those observed for the known highly active antagonists thatserved as lead for the present design, N-methylscopolamine andglycopyrrolate (9.2-9.9 and 8.7-9.9, respectively). As expected, theracemic forms, Compounds (a) and (b), showed lower receptor bindingaffinities than their corresponding 2R isomers (differences significantat p<0.05 level for M₃, t-test or nonparametric Mann-Whitney U test),confirming that stereospecificity is important at these receptors.

TABLE 2 Receptor binding affinities and pA₂ values. Subtypes of clonedmuscarinic receptors ^(a) Compound M₁ M₂ M₃ M₄ pA₂ ^(b) (c) 8.89 ± 0.048.87 ± 0.05 9.00 ± 0.06 9.52 ± 0.01 8.31 ± 0.05 (0.83 ± 0.11) (1.10 ±0.11) (0.83 ± 0.01) (0.83 ± 0.01) (a) 7.91 ± 0.05 7.79 ± 0.11 7.80 ±0.10 8.29 ± 0.19 7.90 ± 0.13 (1.02 ± 0.12) (1.25 ± 0.01) (1.17 ± 0.18)(1.12 ± 0.05) (d) 8.67 ± 0.16 8.84 ± 0.34 8.74 ± 0.02 8.85 ± 0.13 8.55 ±0.16 (0.86 ± 0.08) (0.92 ± 0.01) (1.09 ± 0.15) (0.89 ± 0.02) (b) 7.51 ±0.17 7.32 ± 0.07 7.54 ± 0.15 7.94 ± 0.09 7.36 ± 0.34 (0.91 ± 0.09) (1.23± 0.06) (1.18 ± 0.08) (1.18 ± 0.09) glycopyrrolate 9.76 ± 0.05 9.19 ±0.18 8.73 ± 0.05 9.90 ± 0.08 8.57 ± 0.12 (1.37 ± 0.20) (0.99 ± 0.11)(1.14 ± 0.25) (1.02 ± 0.01) scopolamine 9.69 ± 0.01 9.18 ± 0.21 9.29 ±0.12 9.92 ± 0.21 9.16 ± 0.19 methyl bromide (0.92 ± 0.10) (1.02 ± 0.02)(1.07 ± 0.01) (0.90 ± 0.04) ^(a) Data of the receptor bindingexperiments represent mean ± S.D. of 3 experiments. The numbers inparentheses denote Hill slopes. ^(b) pA₂ values were determined on 4-6ileum strips obtained from different animals. Data represent mean ± SD.pA₂ Studies

The pA₂ values determined from guinea pig ileum contraction assays,which represent the negative logarithm of the molar concentration of theantagonist that produces a two-fold shift to the right in an agonist'sconcentration-response curve, are a classical functional study ofanticholinergic affinity (at M₃ muscarinic receptors). For the softanticholinergics of the present study, the pA₂ values obtained fromileum longitudinal contractions by using carbachol as agonists with themethod of van Rossum (Table 2) were, in general, comparable to thepK_(i) values obtained in the M₃ receptor binding studies. The 2Risomers were again significantly more active than the correspondingracemates, and the most active soft analog [Compound (d),pA₂=8.55_(±0.16)] showed activity similar to glycopyrrolate(pA₂=8.57_(±0.12)).

Mydriatic Activities of Soft Analogs

The mydriatic effects of the new soft derivatives. Compounds (a) and(b), were compared to those of glycopyrrolate in rabbits. Mydriaticresponses were recorded at appropriate time-intervals after theadministration of the drugs as % changes in pupil size. Maximum response(R_(max), % change in pupil size at 1 h after administration) and areaunder the response-time curve (AUC^(eff)) are shown in Table 3. Whereas,there are no significant differences among the R_(max) maximum responsesamong all treatments considered (compounds and concentrations of Table3), there dearly are among the AUC^(eff)s (p<0.05, ANOVA followed byTukey-Kramer multiple comparison test). Glycopyrrolate (0.1%, 0.2%) gavesignificantly longer-lasting effects (larger AUC^(eff)s) than any of thesoft drugs. The soft ethyl compounds seem somewhat more potent thancorresponding methyl analogs, and the 2R isomers seem more potent thanthe isomeric mixtures, In agreement with soft drug design principles,their duration of action is much shorter than that of the “hard”glycopyrrolate as illustrated in FIG. 3A and FIG. 3B forpharmacodynamically equipotent doses. The mydriatic activity of Compound(c), Compound (d), and glycopyrrolate lasted for 24, <48, and 144 h,respectively, indicating that the soft drugs are easily hydrolyzed andrapidly eliminated from the body after the desired pharmacologicaleffect is achieved. In agreement with this and unlike other traditionalanticholinergics, these soft drugs did not induce dilation of the pupilin the contralateral (water-treated) eye, indicating no or just lowsystemic side-effects. Therefore, these compounds are safe, promisingshort acting anticholinergics with the possibility of largely reducedunwanted side effects.

TABLE 3 Maximum response (R_(max), % change in pupil size at 1 h afteradministration) and area under the response-time curve (AUC^(eff)). Dataindicate mean ± SD of four trials. Compound Conc. (%) R_(max) (%)AUC^(eff) _((0-144 h)) (a) 0.5 45.83 ± 4.81  185 ± 35  1 59.58 ± 15.72467 ± 114 (b) 0.5 44.65 ± 13.99 596 ± 274 1 58.33 ± 12.27 645 ± 409 (c)0.5 52.92 ± 13.41 752 ± 342 1 57.08 ± 11.66 875 ± 197 (d) 0.5 53.96 ±13.27 1170 ± 308  1 56.04 ± 11.69 1532 ± 526  glycopyrrolate 0.05 51.46± 7.71  2779 ± 443  0.1 55.83 ± 6.42  4074 ± 459  0.2 56.04 ± 10.10 5047± 1631

In conclusion, a set of new glycopyrrolate-based soft anticholinergicshas been designed, synthesized, and tested. They were found to havereceptor binding affinities comparable to those of glycopyrrolate orN-methylscopolamine, and good, but short-lasting mydriatic activity withno or just minimal systemic effects due to their soft nature that allowseasy, one-step metabolism into a designed-in metabolite after exertingtheir desired pharmacological activity.

Further Studies

Purpose. Because stereospecificity is known to be important atmuscarinic receptors, isomers of both N-substituted softanticholinergics based on glycopyrrolate, (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(alkoxycarbonylmethyl)-1-methylpyrrolidiniumbromide methyl and ethyl esters, Compounds (c) and (d), and theirzwitterionic metabolite, (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-methyl-1-carboxymethylpyrrolidiniuminner salt, were synthesized and their pharmacological activities wereevaluated in vitro and in vivo.

Methods. The isomers of Compounds (c) and (d) were synthesized with bothoptically pure methyl-cyclopentylmandelate and3-hydroxy-N-methylpyrrolidine. Trans-esterification followed byquarternization with alkyl bromoacetate gave four isomers of the methylor ethyl ester with the nitrogen chiral center unresolved. Thehydrolysis of these four isomers followed by HPLC separation resulted ineight fully resolved isomers of the corresponding acid. Thepharmacological activities were assessed using the in vitroreceptor-binding assay, guinea pig ileum pA₂-assay, and in vivo rabbitmydriatic effect. The results were compared with that of conventionalanticholinergic agents such as glycopyrrolate, N-meythylscopolamine, andtropicamide as well as that of previously prepared racemates and 2Risomers.

Results. The receptor binding at cloned human muscarinic receptors(M₁-M₄ subtypes), pK_(i) values, of these newly synthesized methyl andethyl ester isomers were in the 6.0-9.5 range, and zwitterion isomers in5.0-8.6 range. In both cases. 2R isomers were found significantly moreactive than 2S isomers (27-447 times for methyl ester isomers, and 6 to4467 times for zwitterion isomers). Among four isomers of the methylester Compound (c) (with chiral center 1′ unresolved), the 3′R isomerswere more active than the corresponding 3′S isomers (1.5-12.9 times).However, in the case of zwitterion isomers, the 3′S isomers were notalways more active than the corresponding 3′R isomers, indicating thatactivity determined based on chiral center 3′ is significantly affectedby the configuration of other two chiral centers, 2 and 1′. In thecompletely resolved 8 zwitterion isomers (all the three chiral centersresolved), it was found that 1′S isomers were more active than thecorresponding 1′R isomers in all cases (1.8-22.4 times). The resultsalso indicate that some isomers showed good M₃/M₂ muscarinic-receptorsubtype-selectivities (about 3-5 times), and 2R and 3′S were thedetermining configurations for this property. Guinea pig ileum assaysand rabbit mydriasis test on zwitterion isomers double confirmed thestereospecificity. In rabbit eyes, some 2R-zwitterion isomers showedmydriatic potencies similar to glycopyrrolate and exceeded tropicamide,but their mydriatic effects lasted considerably less time, and they didnot induce dilation of the pupil in the contralateral, water-treatedeyes. These results indicate that, in agreement with their soft nature,they are locally active, but safe and have a low potential to causesystemic side effects. The pharmacological potency of eight zwitterionisomers was concluded to be (2R1′S3′S, 2R1′S3′R and2R1′R3′S)>2R1′R3′R>2S1S3′R>(2S1′S3′S and 2S1′R3′R)>2S1′R3′S.

Conclusions. The stereospecificity and M₃1M₂ muscarinic-receptorsubtype-selectivity of soft anti-cholinergics, Compounds (c) and (d) andtheir zwitterionic metabolite, have been demonstrated. Adding to theprevious results, safe use of these soft drugs has been confirmed.

Introduction

Stereospecificity of anticholinergics is important at muscarinicreceptors, Compounds (c) and (d), (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(alkoxycarbonylmethyl)-1-methylpyrrolidiniumbromide, and their common zwitterionic metabolite, (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-methyl-1-carboxymethylpyrrolidiniuminner salt, have shown promising activity and safety in animal studies.These compounds indeed exhibited stereospecificity toward muscarinicreceptors, and the anticholinergic activity has been improved with the2R configuration. In addition, the zwitterionic metabolite also showed amoderate M₃/M₂ muscarinic-receptor subtype-selectivity that indicates areduced systemic cardiac side effect. However, the molecules of thistype of soft analogs possess three chiral centers, so that each racemiccompound may contains up to eight different isomers, that is 2R1′R3′R,2R1′R3′S, 2R1′S3′R, 2R1′S3′S, 2S1′R3′R, 2S1′R3′S, 2S1′S3′R, and 2S1′S3′Sas displayed below:

Thus, the above-described investigations in the soft glycopyrrolateisomers based on one resolved chiral center (2R or 2S) only expressedthat 2R enantiomers (a mixture of four diastereoisomers 2R1′3′R,2R1′R3′S, 2R1′S3′R, 2R1′S3′S) were more active than 2S enantiomers (amixture of 2S1′R3′R, 2S1′R3′S, 2S1′S3′R, 2S1′S3′S). In this section,further investigations in the stereospecificity of these softglycopyrrolates are reported using five partially-resolved softanticholinergics isomers and eight fully resolved zwitterion metabolite,isomers (as described for 2R and 2S enantiomers). The compounds weresystematically synthesized and isomers were separated. The relativepharmacological activities and M₃/M₂ muscarinic-receptorsubtype-selectivities were investigated by in vitro receptor-bindingassay, in vitro guinea pig ileum pA₂-assay, and in vivo mydriatic effectin rabbits.

Materials and Methods

Materials

Glycopyrrolate (glycopyrronium bromide) was kindly provided byBoehringer Ingelheim Chemicals, Inc. Carbamylcholine bromide(carbachol), atropine methylbromide (atropine MeBr), and scopolaminemethylbromide (scopolamine MeBr) were obtained from Sigma Chemicals Co.(St. Louis, Mo.), and tropicamide (1%) was obtained from Bausch & LombPharmaceutical (Tampa, Fla.). N-[³H]-Methyl-scopolamine (NMS) wasobtained from Amersham Biosciences UK Limited (Buckinghamshire, UK).Cloned human muscarinic receptor subtypes M₁-M₄ were obtained fromApplied Cell Science Inc. (Rockville, Md.). Scintiverse BD was fromFisher Scientific Co, (Pittsburgh, Pa.), (R)-3-hydroxy pyrrolidinehydrochloride and (S)-3-hydroxy pyrrolidine hydrochloride were fromAstatech Inc. (Monmouth Junction. NJ), N-[³H]-Methyl-scopolamine (NMS)was from Amersham Biosciences UK Limited (Buckinghamshire, UK). Clonedhuman muscarinic receptor subtypes M₁-M₄ were from Applied Cell ScienceInc, (Rockville, Md.). Scintiverse BD was from Fisher Scientific Co.(Pittsburgh, Pa.). Other chemicals used for synthesis were reagent orHPLC grade, and were obtained from Aldrich (Milwaukee, Wis.) and FisherScientific Co. Melting points were taken on Fisher-Johns meltingapparatus. NMR spectra were recorded on Bruker Advance 300, 400 and 500MHz NMR spectrometers, and are reported in ppm relative to TMS. NOESYwas performed using 2D NMR spectrometer, Mercury-300BB, Animal studieswere performed in accordance with the Guide for the Care and Use ofLaboratory Animals adopted by the National Institute of Health, USA.Institutional animal care and use committee (IACUC) approval wasobtained prior to the initiation of this research and during itsexecution.

Synthesis of 2R-Isomers Racemic Cyclopentylmandelic Acid, 1

Cyclopentylmagnesium bromide ether solution (100 ml, 2M; 0.2 mol) wasadded drop-wise to benzoylformic acid (15 g, 0.1 mol) in 330 ml ofanhydrous ethyl ether at 0° C. The mixture was stirred at 0° C. for 30min and at room temperature for 24 h. The reaction mixture was treatedwith 1 N HCl, and the aqueous solution was extracted with ether. Thecombined ether solution was treated with K₂CO₃ solution. The potassiumcarbonate solution was acidified with HCl and extracted with ethertwice. The ether solution was dried with anhydrous sodium sulfate andevaporated to give a crude product. The crude product was washed withwater to get pure racemic cyclopentylmandelic acid 1 (8.0 g, 36.4%).Needle-like crystal, m.p.: 153-154° C. ¹H NMR (CDCl₃, 300 MHz):1.28-1.39, 1.42-1.50, 1.51-1.61, 1.63-1.72 [8H, m, (CH₂)₄], 2.93 [1H, p,CHC(OH)], 7.26-7.30, 7.33-7.36, 7.65-7.67 (5H, m, Ph) ppm.

Resolution of Racemic Cyclopentylmandelic Acid, R(−)1

(−)-Strychnine (11.4 g) in 100 ml of methanol (suspension) was added toracemic cyclopentylmandelic acid 1 (7.5 g) in methanol (20 ml) at roomtemperature. The reaction solution was allowed to stand overnight. Thecrystals were filtered and crystallized again with hot methanol. Thesecond crop of crystals was collected by filtration and treated withsodium hydroxide solution. The basic solution was extracted withmethylene chloride twice (methylene chloride solution discarded), andthen acidified with hydrochloric acid to recover the resolvedcyclopentylmandelic acid. To this resolved acid (20.6 mg in 0.1 ml ofethyl acetate), 13 μL of (+)-α-phenylethylamine was added. Theprecipitate which formed was washed with hexane three times and driedunder vacuum. The precipitate was identified by NMR as optically purecyclopentylmandelic acid, R(−)1, (2.5 g, 33.3%). M.p.: 121-122° C.[α]^(25°) _(D)=−22.5° (c=1 g/100 ml, CHCl₃). ¹H NMR(CDCl₃, 300 MHz):1.28-1.39, 1.42-1.50, 1.51-1.61, 1.64-1.73 [8H, m, (CH₂)₄], 2.93 [1H, p,CHC(OH)], 7.25-7.28, 7.32-7.35, 7.64-7.65 (5H, m, Ph) ppm.

Methyl (−)-cyclopentylmandelate, R(−)2

To a mixture of (−)-cyclopentylmandelic acid, R(−)1, (1.83 g, 8.3 mmol)and potassium carbonate (2.87 g, 21 mmol) in DMF (21 ml), methyl iodide(3.53 g, 25 mmol) was added at room temperature. The mixture was stirredat room temperature for 2 h, and then poured into water and extractedwith hexane three times. Evaporation of dried hexane extract gave acrude product. Flash chromatography of the crude product on silica gelwith 1.5:1 hexane:methylene chloride gave pure product R(−)2 (1.90 g,95%), ¹H NMR (CDCl₃, 300 MHz): 1.32-1.36, 1.43-1.61 [8H, m, (CH₂)₄],2.90 [1H, p, CHC(OH)], 3.71 (1H, s, OH), 3.79 (3H, s, CH₃), 7.25-7.28,7.31-7.35, 7.63-7.65 (5H, m, Ph) ppm.

(R)-3-Hydroxy-N-Methyl pyrrolidine, (R)3

In a 100 ml flask, 2 g (R)-3-Hydroxy pyrrolidine, 25 ml THE, 0.49 gparaformaldehyde and 1.5 g formic acid (90%) were added. The mixture wasstirred under reflux for 5 hours (until all solid disappeared), thencooled at 0° C. and added with 10 nil of NaOH solution (10 N) to adjustthe pH to about 10. The organic layer was separated and dried overMgSO₄. After filtering the dried solution and removing the solvent(THF), an oily product (1.5 g, 92%) of (R)3 was obtained. ¹H NMR (CDCl₃,300 MHz): 1.50-1.60 (m, 1H), 1.98-2.10 (m, 1H), 2.25 (s, 3H), 2.25-2.40(m, 2H), 2.50-2.60 (m, 1H), 2.61-2.70 (m, 1H), 3.80 (brs, 1H), 4.20-4.30(m, 1H).

(S)-3-Hydroxy-N-Methyl pyrrolidine, (S)3

Synthesis of (S)3 was the same as for (R)3, except that the startingmaterial was (S)-Hydroxy pyrrolidine. The resultant product (S)3 (1.5 g,92%) was also an oil. ¹H NMR (DMSO-D6 300 MHz): 1.50-1.60 (m, 1H),1.98-2.05 (m, 1H), 2.15 (s, 3H), 2.15-2.35 (m, 2H), 2.45-2.52 (m, 1H),2.61-2.70 (m, 1H), 4.20 (brs, 1H), 4.60-4.70 (m, 1H).

3(R)—N-Methyl-3-pyrrolidinyl-2(R)-cyclopentylmandelate, 4

A solution of R(−)2 (0.7 g, 3 mmol) and (R)3 (0.7 g, 7 mmol) in 40 nilof toluene was heated until 20 ml of toluene had distilled,Approximately 0.003 g of sodium was added, and the solution was stirredand heated for 2 h as the distillation was continued. More toluene wasadded at such a rate as to keep the reaction volume constant. Additionalsodium was added at the end of an hour. The solution was then cooled andextracted with 3N HCl. The acid extract was made alkaline withconcentrated NaOH and extracted three times with ether. Removal of driedether solution gave a crude oil. Flash chromatography of the crudeproduct on silica gel with 8:1 of EtOAc and EtOH gave an oil product of4 (0.4 g, 44%). ¹H NMR (CDCl₃, 300 MHz): 1.28-1.37, 1.51-1.70,1.83-1.90[8H, m, (CH₂)₄], 2.27-2.40 (m, 3H), 2.52-2.55 (m, 1H),2.64-2.72 (m, 1H), 2.74-2.81 (m, 1H), 2.33 (3H, s, NCH₃), 2.93 [1H, p,CHC(OH)], 3.85 (1H, bs, OH), 5.22 (m, 1H), 7.24-7.27, 7.31-7.35,7.64-7.66 (5H, m, Ph) ppm.

3(S)—N-Methyl-3-pyrrolidinyl-2(R)-cyclopentylmandelate, 5

Synthesis of 5 was the same as for 4, except (S)3 was used instead of(R)3. The resultant product 5 (0.35 g, 39%) was also an oil. NMR (CDCl₃,400 MHz): 1.28-1.37, 1.51-1.70, 1.75-1.82[8H, m, (CH₂)4], 2.15-2.22 (m,1H), 2.30-2.40 (m, 2H), 2.65-2.70 (m, 1H), 2.70-2.82 (m, 2H, 2.35 (3H,s, NCH₃), 2.95 [1H, p, CHC(OH)], 3.82 (1H, bs, OH), 5.22 (m, 1H),7.24-7.27, 7.31-7.35, 7.64-7.66 (5H, m, Ph) ppm.

(2R,3′R)3-(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide, 6 [Compound (e)]

To Compound 4 (0.3 g, 0.98 mmol) in 12 ml of dry acetonitrile, methylbromoacetate (0.5 g, 3.2 mmol) was added at room temperature. Themixture was stirred for 6 h. Evaporation of acetonitrile gave a crudeproduct. The crude product was dissolved in a small volume of methylenechloride and then poured into 50 ml of dry ethyl ether to precipitate.This step was repeated three times to obtain the pure product 6, orCompound (e), (0.3 g, 70%) as a white powder that was a mixture of twodiastereoisomers in a NMR-estimated ratio of 2:1, ¹H NMR (CDCl₃, 400MHz): 1.30-1.37, 1.41-1.50, 1.55-1.70 [8H, m, (CH₂)₄], 2.10-2.27 (m,1H), 2.79-195 (m, 2H), 3.05, 3.60 (2s, total 3H, N—CH3), 3.75, 3.79 (2s,total 3H, O-Me), 3.95-4.40 (m, 4H), 4.68, 5.16 (2AB, total 2H,N—CH2-COOMe), 5.52-5.58 (m, 1H), 7.23-7.29, 7.31-7.38, 7.56-7.60 (5H, m,Ph) ppm,

(2R,3′S)3-(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide, 7a [Compound (f)]

To Compound 5 (0.16 g, 0.52 mmol) in 8 ml of thy acetonitrile, methylbromoacetate (0.3 g, 1.9 mmol) was added at room temperature. Followingthe same procedure for 6 [Compound (e)] the pure product 7a [Compound(f)] (0.16 g, 80%) was obtained. Compound (f) was also a white powderand a mixture of two diastereoisomers in a NMR-estimated ratio of 2:1.¹H NMR (CDCl₃, 400 MHz): 1.30-1.70 [8H, m, (CH₂)₄], 1.95-2.00, 2.10-2.20(m, 1H), 2.75-2.95 (m, 2H), 3.30, 3.70 (2s, total 3H, N—CH3), 3.78, 3.82(2s, total 3H, 0-Me), 4.00-4.42 (m, 4H), 4.90, 5.38 (2AB, total 2H,N—CH2-COOMe), 5.52-5.58 (m, 1H), 7.23-7.29, 7.31-7.38, 7.56-7.60 (5H, m,Ph) ppm.

(2R,3′S)3-(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide, 7b [Compound (g)]

To Compound 5 (0.16 g, 0.52 mmol) in 10 nil of dry acetonitrile, ethylbromoacetate (0.32 g, 1.9 mmol) was added at room temperature. Themixture was stirred for 22 hours, and the removal of acetonitrile gave acrude product. The crude product was dissolved in small volume ofethylene chloride, and then poured into a 50 ml of dry ethyl ether toafford a precipitate. This procedure was repeated three times, and thepure product 7b, or Compound (g) (0.16 g, 80%) was obtained. Compound(g) was also a white powder and a mixture of two diastereoisomers in aNMR-estimated ratio of 2:1. ¹H NMR (CDCl₃, 400 MHz): 1.32, 1.35 (2t, 3H,CH₃CH₂), 1.40-1.50, 1.53-1.63, 1.65-1.80 [8H, m, (CH₂)₄], 1.93-2.11 (m2H), 2.80-2.96 M, 2H), 3.30, 3.70 (2s, 3H, N—CH3), 4.10-4.60 (m, 6H),4.79, 5.30 (2H, 2 set of dd, CH₂CO₂), 5.53 (1H, m), 7.24-7.29,7.31-7.38, 7.56-7.60 (5H, m, Ph) ppm.

Hydrolysis of Esters

Compounds (e) and (f) were combined with equimolar ratios of 0.1 N NaOH.The mixtures were stirred at room temperature for 3 hours to obtain thecorresponding racemic zwitterionic products, 8 and 9 in aqueous solution(colorless, pH about 6.5). Compound 8 is (2R,3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidiniuminner salt. Compound 9 is (2R,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidiniuminner salt.

HPLC Separations for 8a, 8b, and 9a, 9b

The solutions of 8 and 9 each contained two isomers, 8a, 8b and 9a, 9b,at a ratio of 2:1 that could be separated by HPLC. The HPLC systemconsisted of a Spectra Physics (San Jose, Calif.) SP 8810 isocraticpump, a SP 8450 UV/Vis detector (wavelength set to 230 nm), a SP 4290integrator, and a Supelco Discovery RP Amide C16 column. The mobilephase consisted of acetonitrile and water at a ratio of 30:70. With 100μl injection at a flow rate of 1 ml/min, the retention times were 7.2min for 8a and 9a, and 8.5 min for 8b and 9b. The effluencecorresponding to each isomer was collected, and the solvent wasevaporated to obtain the final zwitterionic isomers, 8a, 8b, and 9a, 9bas following:

(2R,1′R,3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidiniuminner salt, 8a

white powder ¹H NMR (CDCl₃, 300 MHz):1.30-1.65 (m, 8H), 2.02-2.12 (m,1H), 2.20-2.60 (brs, 1H), 2.60-2.80 (m, 1H), 2.82-2.92 (m, 1H), 3.30 (s,3H), 3.55-3.65 (m, 1H), 3.72-3.82 (m, 1H), 3.90-4.05 (m, 2H), 4.10-4.15(m, 1H), 5.38-5.45 (m, 1H), 7.15-7.20 (m, 1H), 7.32-7.38 (m, 2H),7.55-7.62 (m, 2H).

(2R,1′R,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidiniuminner salt, 8b

¹H NMR (CDCl₃, 300 MHz):1.30-1.75 (m, 8H), 2.02-2.10 (m, 1H), 2.10-2.40(brs, 2H), 2.70-2.80 (m, 1H), 2.80-2.90 (m, 1H), 2.95 (s, 3H), 3.55-3.65(m, 2H), 3.85-4.0 (m, 3H), 4.05-4.10 (m, 1H), 5.38-5.45 (m, 1H),7.15-7.20 (m, 1H), 7.25-7.30 (m, 2H), 7.50-7.60 (m, 2H).

(2R,1′R,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl-1-methylpyrrolidiniuminner salt, 9a

white powder, ¹H NMR (CDCl₃, 500 MHz): 1.30-1.65 (n, 8H), 2.02-2.12 (m,1H), 2.50-2.60 (m, III), 2.78-2.88 (m, 1H), 3.25 (s, 31-1), 3.65-4.05(m, 411), 4.15-4.30 (brs, 211), 5.30-5.40 (m, 1H), 7.13-7.23 (m, 1H),7.26-7.32 (m, 2H), 7.55-7.60 (m, 2H).

(2R,1′S,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidiniuminner salt, 9b

white powder, ¹H NMR (CDCl₃, 500 MHz):1.30-1.70 (m, 8H), 1.90-1.98 (m,1H), 2.65-2.70 (m, 1H), 2.85-2.90 (m, 1H), 3.15 (s, 3H), 3.65-3.90 (m,4H), 4.05-4.10 (M, 1H), 4.15-4.22 (brs, 1H), 5.35-5.42 (m, 1H),7.18-7.23 (m, 1H), 7.27-7.32 (m, 2H), 7.53-7.58 (m, 2H).

Determination of Absolute Configurations

Nuclear overhauser effect (NOE) has been used to identify the absoluteconfiguration of the product 8b. Compound was dissolved in CDCl₃, andthe 2D ¹H—¹H spectrum was taken by Mercury-300BB.

Synthesis of 2S-isomersCis-(2S,5S)-2-(tert-butyl)-5-phenyl-1,3-dioxolan-4-one, 10

S(+)-mandelic acid in hexane suspension (50 g, 328 mmol) was added withpivaldehyde (42.7 ml, 396 mmol) then trifluoromethanesulfonic acid (1.23ml, 14 mmol) at room temperature. The mixture was warmed to 36 and thereaction was followed by TLC for 5 hr until no starting material couldbe detected. The mixture was then cooled to room temperature and addedwith 8% aqueous NaHCO₃. The water layer was removed and the organiclayer was dried over Na₂SO₄. After filtration and removal of thesolvent, 62.17 g of the crude product was obtained. Recrystallization ofthe crude product gave 44.71 g of purecis-(2S,5S)-2-(tert-butyl)-5-phenyl-1,3-dioxolan-4-one in 88% yield as aneedle-like crystal. ¹H NMR (CDCl₃, 300 MHz): 1.08 (s, 9H), 5.24 (s,1H), 5.33 (s, 1H), 7.40-7.46 (m, 5H) ppm. ¹³C NMR (CDCl₃, 300 MHz):23.6, 34.4, 77.0, 109.3, 127.0, 128.7, 129.2, 133.4, 147.2.

Cis-(2S,5S)-2-(tert-butyl)-5-phenyl-5-cyclopentyl-1,3-dioxolon-4-one, 11

At −78′C, a lithium bis-(trimethylsilyl)amide in hexane solution (120ml, 120 mmol, 1.0M in hexane) was added to compound 10 (25 g, 113.5mmol, dissolved in 100 ml of dried THF), stirred for 1 hr, followed byaddition of cyclopentyl bromide (25 g, 168 mmol). This reaction was keptat −78° C. for 4 hr, then slowly warmed up to room temperature andcontinued for overnight. The completion of the reaction was followed byTLC. With stirring, a solution of 10% of NH₄Cl (25 ml) was added in themixture. Then, the mixture was poured into a separation funnelcontaining 10% NH₄Cl solution (200 ml), The aqueous layer was discarded,and the organic layer was dried over Na₂SO₄. The solvent was removed togive a crude product, which was then re-crystallized in hexane to give apure product, 11 (20.36 g, yield 63%, white crystal). ¹H NMR (CDCl₃, 300MHz): 1.15 (s, 9H), 1.55-1.95 (m, 8H), 2.74 (m, 1H), 5.62 (s, 1H),7.44-7.56 (m, 3H), 7.88-7.91 (n, 2H) ppm. ¹³C NMR (CDCl₃, 300 MHz):23.5, 24.5, 25.3, 26.6, 35.6, 50.9, 83.2, 110.6, 124.9, 127.5, 127.9,138.9, 173.7.

S(+)-Cyclopentylmandelic Acid, 12

To a solution ofcis-(2S,5S)-2-(tert-butyl)-5-cyclopentyl-5-phenyl-1,3-dioxolan-4-one(14.35 g, 50 mmol) in 100 ml methanol and 50 ml water, 15 g of KOH wasadded slowly. The mixture was stirred and heated (65° C.) to reflux for3-4 hr, then cooled down to the room temperature, and methanol wasremoved. To the aqueous solution, 100 ml of ethyl acetate was added,then acidified to pH 1 with 3N HCl. The mixture was poured into aseparation funnel, and the organic layer was separated. The aqueouslayer was extracted two times with ethyl acetate (50 ml). The combinedorganic layers were dried over Na₂SO₄, filtered, and the solvent wasremoved to provide 13.44 g of yellowish crude product, which wasre-crystallized to give a pure product of S(+)-cyclopentylmandelic acid,12 (6.89 g, yield 62%, white crystal). ¹H NMR (CDCl₃, 300 MHz):1.28-1.75 (m, 8H), 2.94 (m, 1H), 7.24-7.34 (m, 3H), 7.62-7.68 (m, 2H).¹³C NMR (CDCl₃, 300 MHz): 25.9, 26.3, 26.4, 26.9, 47.1, 79.2, 125.8,127.7, 128.2, 140.8, 180.9.

Methyl S(+)-cyclopentylmandelate, 13

S(+)-cyclopentylmandelic acid, 12 (5.5 g, 25 mmol), and potassiumcarbonate (8.61 g, 63 mmol) in DMF (60 ml) solution was added withmethyl iodide (10.6 g, 75 mmol). The mixture was stirred at roomtemperature for 3 hr. poured into water, and extracted with hexane forthree times. Evaporation of dried hexane extract gave a pure product ofS(+)-cyclopentylmandelate, 13 (5.85 g, too %, clear oil). ¹H NMR (CDCl₃,300 MHz): 1.32-1.61 [8H, m, (CH₂)₄], 2.90 [1H, p, CHC(OH)], 3.76 (s,3H), 3.78 (s, 1H), 7.25-7.35 (m, 3H), 7.63-7.65 (m, 2H). ¹³C NMR (CDCl₃,300 MHz): 25.9, 26.2, 26.3, 26.8, 47.1, 53.2, 79.1, 125.8, 127.3, 128.0,141.6, 176.0.

(R)-3-Hydroxy-N-Methylpyrrolidine, (R)3

In a 100 ml flask, 4 g (R)-3-Hydroxy pyrrolidine hydrochloride salt, 50ml THE and 1.3 g NaOH were added and stirred for 20 min. Then, 1.1 gparaforaldehyde and 4.8 g formic acid (90%) were added. The mixture washeated (60° C.) and stirred at reflux for 2 hr until all soliddisappeared. The mixture was cooled to 0° C., combined with 6.5 ml of 10N NaOH solution (pH about 10), and extracted twice by ethyl ether (50ml). The combined organic layer was dried over Na₂SO₄. Evaporation ofthe dried organic layer gave a yellowish, oily product of (R)3 (3.0 g,92%). ¹H NMR (CDCl₃, 300 MHz): 1.65-1.75 (m, 1H), 2.15-2.36 (m, 2H),2.33 (s, 3H), 2.55-2.59 (m, 2H), 2.76-2.85 (m, 1), 4.30-4.40 (m, 1H),4.8-5.10 (brs, 1H). ¹³C NMR (CDCl₃, 300 MHz): 35.4, 41.9, 54.7, 64.9,70.9.

(S)-3-Hydroxy-N-Methylpyrrolidine, (S)3

Synthesis of (S)3 was the same as for (R)3, except the starting materialwas (S)-3-Hydroxypyrrolidine hydrochloride salt. The resultant product(S)3 (3.10 g, 95%) was also an oil. ¹H NMR (CDCl₃, 300 MHz): 1.50-1.60(m, 1H), 2.05-2.30 On, 2H), 2.28 (s, 3H), 2.40-2.50 (m, 2H), 2.70-2.80(m, 1H), 4.25-4.30 (m, 1H), 4.80 (brs, 1H). ¹³C NMR (CDCl₃, 300MHz):35.4, 41.9, 54.7, 64.9, 70.9.

(3R) N-Methyl-3-pyrrolidinyl-(S)-cyclopentylmandelate, 14

In a 250 ml 3-neck flask equipped with Dean-Stark condenser, a mixtureof methyl S(+)-cyclopentylmandelate, 13 (2 g, 8.8 mmol),(R)-3-hydroxy-N-methylpyrrolidine, (R)-3 (2 g, 20 mmol), and 100 ml ofheptane was stirred and heated (110° C.) until 20 ml of heptane had beendistilled. The temperature was reduced to 25″C, and approximately 0.003g of sodium was added. The mixture was stirred and heated to 110° C.again for 3 hr as the distillation was continued. An additional piece ofsodium (0.002 g) was added at the 1 hr point. More heptane was added atsuch a rate as to keep the reaction volume constant. The mixture wascooled to 0° C., mixed with 5 ml of water, and the organic layer wasseparated. The organic layer was extracted with 3N HCl. The acid extractwas made alkaline (pH 10) with 5N NaOH and extracted three times withether. Removal of dried ether solution (over Na₂SO₄) gave a clear, oilyproduct 14 (1.6 g, 61.5%). ¹H NMR (CDCl₃, 300 MHz): 1.28-1.80 [m, 9H],2.15-2.25 (m, 1H), 2.30-2.40 (m, 1H), 2.37 (s, 3H), 2.65-2.80 (m, 3H),2.90-3.00 (m, 1H), 3.85 (1H, brs, OH), 5.22 (m, 1H), 7.20-7.35 (m, 3H),7.64-7.70 (m, 2H). ¹³C NMR (CDCl₃, 300 MHz):26.0, 26.4, 26.5, 26.7,32.1, 42.0, 47.1, 54.8, 62.0, 76.5, 79.1, 125.8, 127.3, 128.0, 141.7,175.3.

(3S) N-Methyl-3-pyrrolidinyl-(S)-cyclopentylmandelate, 15

Following the same procedure as for 14, except (S)-3 was used instead of(R)-3, a clear, oily product of 15 (2.33 g, 89.6%) was obtained. ¹H NMR(CDCl₃, 300 MHz): 1.24-1.70 (m, 9H), 1.80-1.88 (m, 1H), 2.25-2.40 (m,2H), 2.35 (s, 3H), 2.55-2.70 (m, 2H), 2.75-2.82 (m, 1H), 2.90-3.00 (m,1H), 3.95 (1H, m, OH), 5.22 (m, 1H), 7.24-7.40 (m, 2H), 7.64-7.69 (m,5H). ¹³C NMR (CDCl₃, 300 MHz):26.0, 26.3, 26.4, 26.7, 32.6, 42.0, 47.1,54.9, 61.6, 76.4, 79.2, 125.8, 127.3, 128.0, 141.7, 175.2.

(2S,3′R)3-(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide, 16 [Compound (h)]

Compound 14 (0.6 g, 1.96 mmol) in 30 ml of dry acetonitrile was combinedwith methyl bromoacetate (1.0 g, 6.4 mmol) at room temperature. Themixture was stirred for 3 hr. Evaporation of acetonitrile gave a crudeproduct. The crude product was dissolved in a small volume of methylenechloride and poured into a 100 ml of dry ethyl ether to obtain aprecipitate. This procedure was repeated three times and gave compound(h) as the product (0.81 g, 89%, white powder). ¹H NMR (CDCl₃, 300 MHz):1.30-1.70 (m, 8H), 1.82-1.95 (brs, 1H), 2.10-2.20 (m, 1H), 2.75-2.90 (m,2H), 3.25, 3.60 (2s, total 3H, N—CH3), 3.75, 3.79 (2s, total 3H, O-Me),4.10-4.60 (m, 4H), 4.92, 5.35 (2AB, total 2H, N—CH2-COOMe), 5.52-5.58(m, 1H), 7.23-7.38 (m, 3H), 7.56-7.60 (m, 2H). ¹³C NMR (CDCl₃, 300 MHz);25.8, 25.9; 26.3, 26.4; 26.4, 26.5; 27.0, 27.0; 29.8, 30.1; 45.9, 46.8;50.2, 51.4; 53.2, 53.2; 62.2, 63.2; 64.6, 64.7; 69.6, 69.7; 72.8, 73.1;79.4, 79.6; 125.7, 125.7; 127.6, 127.9; 128.2, 128.4; 141.0, 141.2;165.3, 165.5; 173.9, 174.2.

(2S,3′S)3-(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-methoxycarbonylmethyl-1-methylpyrrolidiniumbromide, 17 [Compound (i)]

Following the same procedure as for Compound (h), except compound 15 wasused instead of compound 14, the product Compound (i) (0.8 g, 88%, whitepowder) was obtained. ¹H NMR (CDCl₃, 300 MHz): 1.30-1.75 (m, 8H),1.80-1.90 (brs, 1H), 2.15-2.30 (m, 1H), 2.78-2.95 (m, 2H), 3.10, 3.65(2s, total 3H, N—CH3), 3.75, 3.78 (2s, total 3H, O-Me), 4.15-4.52 (m,4H), 4.85, 5.38 (2AB, total 2H, N—CH2-COOMe), 5.50-5.58 (m, 1H),7.23-7.38 (m, 3H), 7.56-7.66 (m, 2H). ¹³C NMR (CDCl₃, 300 MHz): 25.8,25.9; 26.2, 26.3; 26.3, 26.4; 26.8, 26.9; 29.4, 29.6; 45.6, 46.9; 50.1,51.4; 53.1, 53.1; 62.2, 63.3; 64.8, 64.8; 69.5, 69.8; 72.8, 73.2; 79.4,79.6; 125.6, 125.9; 127.6, 127.9; 128.2, 128.4; 140.7, 141.1; 165.2,165.5; 173.9, 174.2.

Hydrolysis of Esters & HPLC Separations

The procedures used for obtaining the 2S-isomers 18a, 18b, 19a and 19b(white powder) were the same as thr 2R-isomers 8a, 8b, 9a and 9b.

(2S,1R,3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidiniuminner salt. 18a

¹H NMR (CDCl₃, 300 MHz):1.30-1.65 (m, 8H), 2.02-2.45 (m, 2H), 2.82-2.90(m, 1H), 3.10-3.18 (m, 1H), 3.25 (s, 3H), 3.50-4.05 (m, 6H), 5.34-5.40(m, 1H), 7.23-7.38 (m, 3H), 7.50-7.68 (m, 2H).

(2S,1′S,3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethy)-1-methylpyrrolidiniuminner salt, 18b

¹H NMR (CDCl3, 300 MHz):1.45-1.85 (m, 911), 2.05-2.15 (m, 1H), 2.80-2.90(m, 1H), 3.00-3.10 (m, 1H), 3.35 (s, 3H), 3.70-3.80 (m, 1H), 3.90-4.10(m, 4H), 4.22-4.35 (m, 1H), 5.50-5.60 (m, 1H), 7.36-7.55 (m, 311),7.72-7.80 (m, 2H).

(2S,1′R,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidiniuminner salt, 19a

¹H NMR (CDCl3, 300 MHz): 1.20-1.65 (m, 8H), 1.95-2.10 (m, 1H), 2.20(brs, 1H), 2.40-2.50 (m, 1H), 2.78-2.90 (m, 1H), 3.15 (s, 3H), 3.70-3.90(m, 2H), 3.96-4.20 (m, 4H), 5.38-5.50 (m, 1H), 7.20-7.38 (m, 3H),7.55-7.65 (m, 2H).

(2S,1′S,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidiniuminner salt, 19b

¹H NMR (CDCl3, 300 MHz): 1.35-1.70 (m, 8H), 2.00-2.15 (m, 1H), 2.70-2.90(m, 2H), 3.00 (s, 3H), 3.42 (brs, 1H), 3.58-3.68 (m, 2H), 3.80-3.95 (m,3H), 4.08-4.18 (m, 1H), 5.38-5.48 (m, 1H), 7.20-7.40 (m, 3H), 7.55-7.62(m, 2H).

Receptor Binding Affinity

Receptor binding studies on soft anticholinergics isomers and theirzwitterionic metabolite isomers, as well as glycopyrrolate, andN-methylscopolamine were performed with N-[³H]-methyl-scopolamine (NMS)in assay buffer (phosphate-buffered saline, PBS, without Ca⁺⁺ or Mg⁺⁺,pH 7.4), following the protocol from Applied Cell Science Inc.(Rockville, Md.). A 10 mM NaF solution was added to the buffer as anesterase inhibitor. The assay mixture (0.2 mL) contained 20 μL dilutedreceptor membranes (receptor proteins: M₁, 38 μg/mL; M₂, 55 μg/mL; M₃,27 μg/mL; M₄, 84 μg/mL). The final concentration of NMS for the bindingstudies was 0.5 nM. Specific binding was defined as the difference in[³H]NMS binding in the absence and presence of 5 μM atropine for M₁ andM₂ or 1 μM atropine for M₃ and M₄. Incubation was carried out at roomtemperature for 2 hr. The assay was terminated by filtration through aWhatinan GF/C filter (presoaked overnight with 0.5% polyethyleneimine).The filter was then washed six times with 1 mL ice cold buffer (50 mMTris-HCl, pH 7.8, 0.9% NaCl), transferred to vials, and 5 mL ofScintiverse was added. Detection was performed on a Packard 31800 liquidscintillation analyzer (Packard Instrument Inc., Downer Grove, Ill.).Data obtained from the binding experiments were fitted to the equation%[³H] NMS bound=100−[100x^(n)/k(1+x^(n)/k)], to obtain the Hillcoefficient n, and then to the equation %[³H] NMSbound=100−[100x^(n)/IC₅₀/(1+x^(n)/IC₅₀)], to obtain the IC₅₀ values (xbeing the concentration of the tested compound). Based on the method ofCheng and Prusoff (8), K_(i) was derived from the equationK_(i)═IC₅₀/(1+L/K_(d)), where L is the concentration of the radioligand.IC₅₀ represents the concentration of the drug causing 50% inhibition ofspecific radioligand binding, and K_(d) represents the dissociationconstant of the radioligand receptor complex. Data were analyzed by anon-linear least-square curve-fitting procedure using Scientist software(MicroMath Inc., Salt Lake City, Utah).

Determination of pA₂ Values

Male guinea pigs weighing about 400 g were obtained from Harlan Inc.(Indianapolis, Ind.) and fasted overnight. Animals were sacrificed bydecapitation, and the ileum (the region of 5 cm upward of the cecum) wasisolated and removed, The ileum was cut into 2.5 cm pieces and suspendedin an organ bath containing 30 mL of mixture of Tyrode's solution and0.1 mM hexamethonium bromide. The organ bath was constantly aerated withoxygen and kept at 37° C. One end of the ileum strip was attached to afixed support at the bottom of the organ bath, and the other end to anisometric force transducer (Model TRN001, Kent Scientific Corp., Conn.)operated at 2-10 g range. The ileum strip was kept at a 2 g tension, andcarbachol was used as antagonist. The ileum contracted cumulatively uponthe addition of consecutive doses of carbachol (10-20 μL of2×10⁻⁴-2×10⁻³ M in water solution). Contractions were recorded on aphysiograph (Kipp & Zonen Flarbed Recorder, Holland). After the maximumresponse was achieved, the ileum was washed three times, and a freshTyrode's solution containing appropriate concentration of the antagonist(anticholinergic compound tested) was replaced. An equilibration time of10 min was allowed for the antagonists before the addition of carbachol,In each experiment, 5 to 6 different concentrations were used, and aSchild plot was used to obtain the pA₂ values. Four trials wereperformed for each antagonist.

In Vivo Mydriatic Studies

The mydriatic effects of eight completely resolved zwitterionic isomerswere compared to those of glycopyrrolate, tropicamide, (±)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidiniuminner salt [(±)-GA] and (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidiniuminner salt [(2R)-GA] in rabbit eyes. Four healthy, male New-Zealandwhite rabbits weighting about 3.5 kg were used. 100 μL of compound inwater solution (pH 6.5) at various concentrations were administered inthe eyes. Compound solutions were applied to one eye, and water wasapplied to the other eye that served as control. Experiments werecarried out in a light- and temperature-controlled room. At appropriatetime intervals, the pupil diameters of both eyes were recorded. Per centdifference in pupil diameters between each time-point and zerotime-point were calculated for both treated and control eyes andreported as mydriatic responses. Control eye dilations were monitored todetermine whether systemic absorption had occurred or riot. The areaunder the mydriatic response-time curve (AUC^(eff)) was calculated bythe trapezoidal rule, and it was used to compare the activity andduration of action of the tested compounds.

Statistical Analysis

Receptor binding affinities and pA₂ values were compared using studentt-tests. Mydriatic activities (maximum response Rmax % and area underthe effect curves AUC_(eff)) were compared using ANOVA. A significancelevel of P<0.05 was used in all cases.

RESULTS AND DISCUSSION

Synthesis

Five soft anticholinergic ester isomers and eight zwitterionicmetabolite acid isomers were newly synthesized. The 2R diastereoisomers[Compounds (e), (f), (g), 8a, 8b, 9a and 9b] were obtained by thesynthetic pathways described below.

As shown in Scheme 2, first the racemic cyclopentylmandelic acid 1 wassynthesized with cyclopentylmagnesium bromide and benzoylformic acid.This racemic acid was resolved by repeated crystallization of the saltsproduced between this acid and (−)-strychnine. The left rotatory(−22.5″) optically pure free acid R(−)1 was recovered by basification ofthe salts with sodium hydroxide solution followed by acidification withhydrochloric acid. Methylation of R(−)1 with methyl iodide and potassiumcarbonate in DMF at room temperature yields methyl 2R(—)cyclopentylmandelate, R(−)2. Transesterfication of R(−)2 withR-3-hydroxy-N-methylpyrrolidine, (R)-3 (made from R-3-hydroxypyrrolidinewith paraformaldehyde and formic acid), gave(3R)—N-methyl-3-pyrrolidinyl-2R-cyclopentylmandelate 4; or withS-3-hydroxy-N-methylpyrrolidine (S)-3 (made from S-3-hydroxypyrrolidinewith paraformaldehyde and formic acid), gave(3S)—N-methyl-3-pyrrolidinyl-2R-cyclopentyl mandelate 5. Quarternizationof 4 and 5 with methyl or ethyl bromoacetate in acetonitrile gave 6[Compound (e)], 7a [Compound W], and 7b [Compound (g)]. Each of thesehas two diastereoisomers, due to the nitrogen chiral center, with aratio of 2 to 1 (R:S=2:1) that was shown in 1H NMR spectra. Hydrolysisof 6 [Compound (e)] and 7a [Compound (f)] gave their zwitterionic innersalts 8 and 9. Each zwitterionic salt also possesses twodiastereoisomers with a ratio of 2 to 1 that could be separated by HPLCto give zwitterionic isomers 8a, 8b & 9a and 9b. From 1H MAR, 8a, 8b,and 9a, 9b were evidenced to be pairs of diastereoisomers based onchiral nitrogen. To identify the absolute configuration of theseisomers, 8b was chosen and dissolved in CDCl₃ for the investigation ofnuclear overhauser effect (NOE). The 2D ¹H—¹H NOESY spectrum showed thatthe methyl group on the nitrogen was at the same side as the hydrogen atthe 3-position of pyrrolidinium ring. Accordingly, the configuration ofthe nitrogen should be the S form, and the absolute stereochemistry of8b was proved to be (2R,1′R,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidiniuminner salt. Therefore, 8a was (2R,1′R,3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidiniuminner salt; 9a was (2R,1′R,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidiniuminner salt; and 9b was (2R,1′S,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidiniuminner salt.

Grover and coworkers previously reported [J. Org. Chem. 65: 6283-6287(2000)] the highly stereoselective synthesis of (S)-cyclopentylmandelicacid in five steps starting with (S)-mandelic acid. Modification oftheir procedure afforded pure S(+)-cyclopentylmandelic acid in threesteps with good yield. As depicted in Scheme 3, reaction ofS(+)-mandelic acid with pivaldehyde in the presence of the catalysttrifluoromethanesulfonic acid gave the product ofcis-(2S,5S)-2-(tert-butyl)-5-phenyl-1,3-dioxolan-4-one, 10, in about 90%yield. At −78° C., deprotonation of 10 with lithiumbis(trimethylsilyl)amide followed by adding cyclopentyl bromidegeneratedcis-(2S,5S)-2-(tert-butyl)-5-cyclopentyl-5-phenyl-1,3-dioxolan-4-one,11. Base hydrolysis of 11 with potassium hydroxide, followed byacidification with hydrochloric acid provided the expected(S)-(+)-cyclopentylmandelic acid 12. After this step, the sameprocedures as for 8a, 8b, 9a and 9b including methylation,esterification, quaternization and hydrolyses were followed to give thefinal four zwitterionic isomers, (2S,1′R,3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidiniuminner salt 18a [2S1′R3′R-GA]; (2S,1′S,3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidiniuminner salt 1.8b [2S1′S3′R-GA]; (2S,1′R,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidiniuminner salt 19a [2S1′R3′S-GA]; and (2S,1′S,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyrrolidiniuminner salt 19b [2S1′S3′S-GA]. They were also characterized by NMR.

Receptor Binding Studies The receptor binding affinities of softanalogs, pK_(i), determined by radioligand binding assays using humancloned muscarinic receptor subtypes, M₁-M₄, are presented in Table 4.

TABLE 4 Receptor binding affinities, M₃/M₂ selectivities, and pA₂values. Subtypes of cloned muscarinic receptors ^(a) Selectivity ^(b)Compound M₁ M₂ M₃ M₄ M₃/M₂ pA₂ ^(c) (a) ^(d) 7.91 ± 0.05 7.79 ± 0.117.80 ± 0.10 8.29 ± 0.19 1.0 ± 0.0 7.90 ± 0.13 (1.02 ± 0.12) (1.25 ±0.08) (1.17 ± 0.18) (1.12 ± 0.05) (b) ^(d) 7.51 ± 0.17 7.32 ± 0.07 7.54± 0.15 7.94 ± 0.09 1.8 ± 0.7 7.36 ± 0.34 (0.91 ± 0.09) (1.23 ± 0.06)(1.18 ± 0.08) (1.18 ± 0.09) (±)-GA ^(d) 6.19 ± 0.06 5.48 ± 0.13 5.84 ±0.07 6.44 ± 0.06 2.4 ± 0.7 6.42 ± 0.30 (1.11 ± 0.06) (1.02 ± 0.20) (1.01± 0.07) (0.84 ± 0.06) (c) ^(e) 8.89 ± 0.04 8.87 ± 0.05 9.00 ± 0.06 9.52± 0.01 1.4 ± 0.2 8.31 ± 0.05 (0.83 ± 0.11) (1.10 ± 0.11) (0.83 ± 0.01)(0.83 ± 0.01) (d) ^(e) 8.67 ± 0.16 8.84 ± 0.34 8.74 ± 0.02 8.85 ± 0.131.1 ± 1.1 8.55 ± 0.16 (0.86 ± 0.08) (0.92 ± 0.01) (1.09 ± 0.15) (0.89 ±0.02) 2R-GA ^(e) 8.11 ± 0.16 7.48 ± 0.12 8.12 ± 0.10 8.23 ± 0.12 4.4 ±0.3 7.20 ± 0.19 (1.12 ± 0.25) (0.95 ± 0.11) (0.80 ± 0.01) (1.02 ± 0.10)(e) ^(f) 8.99 ± 0.04 9.01 ± 0.06 9.06 ± 0.14 9.45 ± 0.01 1.1 ± 0.1 —(1.19 ± 0.12) (1.03 ± 0.09) (1.03 ± 0.18) (1.52 ± 0.66) (f) ^(f) 8.50 ±0.03 7.90 ± 0.04 8.60 ± 0.09 8.87 ± 0.09 5.0 ± 1.1   — ^(h) (1.30 ±0.20) (1.07 ± 0.17) (1.04 ± 0.27) (1.08 ± 0.01) (h) ^(f) 7.23 ± 0.017.22 ± 0.03 6.99 ± 0.08 7.57 ± 0.01 0.6 ± 0.1 — (0.98 ± 0.06) (1.09 ±0.18) (1.15 ± 0.13) (1.11 ± 0.03) (i) ^(f) 6.40 ± 0.05 6.47 ± 0.08 5.95± 0.02 6.39 ± 0.01 0.3 ± 0.0 — (0.92 ± 0.09) (0.99 ± 0.16) (1.06 ± 0.03)(1.44 ± 0.75) (j) ^(f) 8.68 ± 0.11 8.21 ± 0.10 8.64 ± 0.07 8.71 ± 0.382.8 ± 0.8 — (1.21 ± 0.33) (1.27 ± 0.11) (1.33 ± 0.16) (1.15 ± 0.03)  8a^(g) 7.04 ± 0.09 6.43 ± 0.07 6.95 ± 0.04 7.00 ± 0.05 3.5 ± 0.2 6.32 ±0.23 (0.97 ± 0.13) (0.85 ± 0.21) (1.06 ± 0.04) (0.93 ± 0.01)  8b ^(g)8.13 ± 0.06 7.63 ± 0.02 8.15 ± 0.02 8.33 ± 0.04 3.3 ± 0.0 7.45 ± 0.21(1.25 ± 0.01) (0.82 ± 0.15) (0.84 ± 0.17) (1.00 ± 0.06)  9a ^(g) 7.98 ±0.01 7.39 ± 0.09 8.04 ± 0.01 8.15 ± 0.06 5.2 ± 0.7 7.33 ± 0.28 (1.02 ±0.03) (0.80 ± 0.22) (0.96 ± 0.03) (1.01 ± 0.06)  9b ^(g) 8.32 ± 0.047.64 ± 0.01 8.46 ± 0.12 8.56 ± 0.07 5.5 ± 1.1 7.15 ± 0.12 (1.01 ± 0.01)(1.00 ± 0.04) (0.80 ± 0.21) (0.86 ± 0.06) 18a ^(g) 5.87 ± 0.04 5.65 ±0.06 5.54 ± 0.16 5.79 ± 0.12 0.8 ± 0.1 5.14 ± 0.38 (1.06 ± 0.05) (1.24 ±0.07) (1.02 ± 0.12) (0.88 ± 0.04) 18b ^(g) 6.67 ± 0.06 6.35 ± 0.01 6.22± 0.05 6.47 ± 0.01 0.7 ± 0.0 5.69 ± 0.13 (1.08 ± 0.03) (1.01 ± 0.01)(1.04 ± 0.08) (1.30 ± 0.28) 19a ^(g) <4.5 <4.5 <4.5 <4.5 — <4 — — — —19b ^(g) 5.84 ± 0.06 5.61 ± 0.00 5.61 ± 0.09 5.85 ± 0.05 1.0 ± 0.2 5.03± 0.26 (1.13 ± 0.10) (1.20 ± 0.02) (1.03 ± 0.01) (0.95 ± 0.18)glycopyrrolate 9.76 ± 0.05 9.19 ± 0.18 8.73 ± 0.05 9.90 ± 0.08 0.4 ± 0.28.57 ± 0.12 (1.37 ± 0.20) (0.99 ± 0.11) (1.14 ± 0.25) (1.02 ± 0.01)scopolamine 9.69 ± 0.01 9.18 ± 0.21 9.29 ± 0.12 9.92 ± 0.21 1.3 ± 0.49.16 ± 0.19 methyl bromide (0.92 ± 0.10) (1.02 ± 0.02) (1.07 ± 0.01)(0.90 ± 0.04) ^(a) Receptor binding at cloned human muscarinic receptors(M₁-M₄ subtypes); pK_(i) data represent mean ± SD of 3 experiments, andthe numbers in parentheses denote Hill slopes. ^(b) M₃/M₂ affinity ratio(times) ^(c) pA₂ values were determined on 4-6 ileum strips obtainedfrom different animals, and data represent mean ± SD. ^(d) Racemicforms. ^(e) Isomers based on the chiral center 2. ^(f) Isomers based onthe chiral centers 2 & 3′. ^(g) Isomers based on the chiral centers 2,3′, & 1′. ^(h) Data not available or not detectable.

The pK_(i) of newly synthesized isomers were compared with that of theracemic and 2R isomeric parent soft drugs [the methyl ester Compound (c)and the ethyl ester Compound (d)], racemic and 2R isomeric GA (thezwitterionic metabolite) as well as those of glycopyrrolate andN-methylscopolamine. pK_(i) of the racemic forms, Compound (a) andCompound (b), showed lower receptor binding affinities than theircorresponding 2R isomers (7.8-8.3 vs. 8.7-9.5), confirming thatstereospecificity is important at these receptors. The potencies ofthese 2R isomers are similar to those of glycopyrrolate (8.7-9.9) andN-methylscopolamine (9.2-9.9). Resolution of 2 and 3′ chiral centers ofracemic Compound (a) resulted in four stereoisomers, Compounds (e), (t),(h) and (i) with pK_(i) values of 9.0-9.5, 7.9-8.9, 7.0-7.6 and 6.0-6.5,respectively. These numbers indicate that among the methyl esterisomers, not only 2R isomers are more potent than the corresponding 2Sisomers, but also that 3′R isomers are more potent than 3′S isomers. The2R3′S isomer of the ethyl ester, Compound (j), showed a pK_(i) value of8.2-8.7, the same as the 2R3′S isomer of the methyl ester, In the sametable, the M₃/M₂ muscarinic-receptor subtype-selectivities were alsocalculated. Contrary to the previously reported 2R isomer of the methyland ethyl esters, Compounds (c) and (d), that show no M₃1M₂ subtypeselectivity, the 2R3′S isomers of the methyl and ethyl esters, Compounds(e) and (j), show significantly increased M₃/M₂ muscarinic-receptorsubtype-selectivity (p<0.01, t-test assuming equal variances). The M₃affinity was 5.0±1.1 times of M₂ affinity in the case of Compound (e),and 2.8±0.8 times in the case of Compound (j). This indicates that theconfiguration of chiral center 3′ may play an important role in thesafety profile of this type of soft anticholinergics.

The receptor-binding pK_(i) of racemic (±) GA and isomeric 2R-GAobtained earlier are also shown in Table 4. In agreement with soft drugdesign principles that the acidic moiety formed by hydrolysis of theparent soft drug ester inactivates the drug, the zwitterions were foundconsiderably less active than their corresponding parent esters, e.g.pK_(i) of (±)GA, 5.5-6.4, vs. Compound (a), 7.8-8.3, and Compound (b),7.3-7.9; and pK_(i) of 2R-GA, 7.5-8.2, vs. Compound (c), 8.9-9.5, andCompound (d), 8.7-8.9 (3-4). As discussed previously, the zwitterionicmetabolite retains some activity because the electronic distribution inits structures somewhat resembles those of the neutral, activeanticholinergics, In this study, to obtain a better picture of thestereospecificity/stereoselectivity of this type of anticholinergic, thezwitterionic form was chosen as a model compound for the investigation,since the zwitterion GA, either in its racemic or its 2R isomeric form,was very soluble and stable in aqueous solutions (buffer or biologicalmedia, pH 6-8). In addition, 2R-GA has been found active at topicalsites (e.g. in rabbit eyes), and could be excreted unchanged, rapidlythrough urine (t_(1/2) 10-15 min after i.v. in rats). In Table 4, thepK_(i) of the completely resolved eight isomers of ±GA, 2R1′R3S-GA,2R1′S3′R-GA, 2R1′R3′S-GA, 2R1′S3′S-GA, 2S1R3′R-GA, 2S1′S3R-GA,2S1′R3′S-GA, 2S1′S3′S-GA was in a wide range of 4.5-8.6. In all cases,the 2R isomers are more potent than the 25 isomers, and the 1′S isomersare more potent than the 1′R isomers, The comparative potencies for 3′Rand 3′S isomers varied depending on the configuration of chiral center2, e.g. 2R1′R3′S>2R1′R3′R and 2R1′S3′S>2R1′S3′R; but 2S1′R3′R> 2S1′R3′Sand 2S1′S3′R>2S1′S3′S. Also, the same as previous methyl ester isomers,among 2R isomers of the acid, the 2R3′S isomers (2R1R3′S and 2R1′S3′S)showed highest M₃/M₂ muscarinic-receptor subtype-selectivities (5.2-5.5times) followed by the 2R3′R isomers (2R1′R3′R and 2R1′S3′R, 3.3-3.5times), The 2S isomers did not show any M₃/M₂ selectivity. Thus, theimportance of the chiral center 2 and 3′ configuration (2R3′S) on theM₃/M₂ selectivity of this type of anticholinergics has beendemonstrated.

In order to show the comparative stereoselectivity (times) based on eachchiral center, the ratio of binding activities of each correspondingpaired isomers was calculated, and the results are shown in Table 5, Theresults displayed are comparative potencies (times) calculated from thereceptor binding affinities, pK_(i), in Table 4. The difference inreceptor binding affinities between 2R and 2S isomers is significant (27to 447 times for the methyl ester isomers, and 6 to 4467 times forzwitterion isomers). The 3′R isomers of the methyl ester (with chiralcenter I unresolved, 2R3′R & 2S3′R methyl esters) are more active (1.5to 12.9 times) than their corresponding 3′S isomers (2R3′S & 2S3′Smethyl esters). However, in the acid, the 3′S isomers were not alwaysmore active than the corresponding 3′R isomers, e.g. in 2R isomers,3′S>3′R (2R1′R3′S>2R1′R3′R and 2R1′S3′S>2R1′S3′R) but in 2S isomers,3′R>3′S (2S1′R3′R>2S1′R3′S and 2S1′S3R>2S1′S3′S). Also, there are moresignificant differences between 2R1′R3′S and 2R1′R3′R than between2R1′S′3′S and 2R1′S3′R (8.7 to 14.1 times vs. 1.0 to 2.0 times), andbetween 2S1′R3′R and 2S1′R3′S than between 2S1′S3′R and 2S1′S3′S (11.0to 23.4 times vs. 4.1 to 6.8 times). These results indicate that theactivity based on chiral center 3′ can be affected by the configurationof the other two chiral centers, 2 and 1′. When comparing all eightzwitterion isomers (with all three chiral centers resolved), it clearlyshows that 1′S isomers were more active than the corresponding 1′Risomers in all cases (1.8-22.4 times).

TABLE 5 Comparative stereoselectivities^(a) Subtypes of clonedmuscarinic receptors^(b) Descrip- Compound M₁ M₂ M₃ M₄ tion^(f) MethylEsters 2R3′S/ 125.9 26.9 446.7 302.0 2R > 2S 2S3′S^(c) 2R3′R/ 57.5 61.7117.5 75.9 2S3′R^(c) 2R3′R/ 3.1 12.9 2.9 3.8 3R > 3S 2R3′S^(d) 2S3′R/6.8 5.6 11.0 1.5 2S3′S^(d) Zwitterions 2R1′R3′R/ 14.8 6.0 25.7 16.22R >> 2S 2S1′R3′R^(c) 2R1′S3′R/ 28.8 19.1 85.1 72.4 2S1′S3′R^(c)2R1′R3′S/ 3020.0 776.2 3467.4 4466.8 2S1′R3′S^(c) 2R1′S3′S/ 302.0 107.2707.9 512.9 2S1′S3′S^(c) 2R1′R3′S/ 8.7 9.1 12.3 14.1 3S > 3R2R1′R3′R^(d) 2R1′S3′S/ 1.5 1.0 2.0 1.7 2R1′S3′R^(d) 2S1′R3′R/ 23.4 14.111.0 19.5 3R > 3S 2S1′R3′S^(d) 2S1′S3′R/ 6.8 5.5 4.1 4.2 2S1′S3′S^(d)2R1′S3′R/ 12.3 15.8 15.8 21.4 1R < 1S 2R1′R3′R^(e) 2R1′S3′S/ 2.2 1.8 2.62.6 2R1′R3′S^(e) 2S1′S3′R/ 6.3 5.0 5.2 4.8 2S1′R3′R^(e) 2S1′S3′S/ 21.912.9 12.9 22.4 2S1′R3′S^(e) ^(a)Affinity ratio (times) between each twoisomers based on each of the three different chiral centers.^(b)Receptor binding at cloned human muscarinic receptors (M₁-M₄subtypes) ^(c)Affinity ratio based on the chiral center 2. ^(d)Affinityratio based on the chiral center 3. ^(e)Affinity ratio based on thechiral center 1. ^(f)Concluded stereoselectivities

In all cases, the Hill coefficients (n) were not very different fromunity indicating that, in general, drug-receptor interactions obeyed thelaw of action and binding took place at only one site.

pA₂ Studies

The pA₂ values determined from guinea pig ileum contraction assays,which represent the negative logarithm of the molar concentration of theantagonist that produces a two-fold shift to the right in an agonist'sconcentration-response curve, are a classical functional study ofanticholinergic affinity (at M₃ muscarinic receptors). For the softanticholinergics of the present study, the pA₂ values obtained fromileum longitudinal contractions by using carbachol as agonist with themethod of van Rossum [Arch. Int. Pharcodyn. 143: 299-330(1963)] arepresented in Table 4. The pill values are in general, comparable to thepK_(i) values obtained in the M₃ receptor binding studies. The pA₂values of newly developed zwitterionic isomers significantly differedbetween 2R and 2S configurations (6.32 to 7.45 and <4 to 5.69,respectively, p<0.01, t-test assuming equal variances). Similar to theabove reported 2R isomer (2R-GA), the pA₂ values of completely resolved2R isomers (2R1′R3′R-GA, 2R1′S3′R-GA, 2R1′R3′S-GA, and 2R1′S3′S-GA) are1 to 2 less than those of the corresponding 2R ethyl and methyl parentester soft drugs, indicating a one to two order of magnitude lessactivity of these zwitterionic compounds. The retained moderate activityof some zwitterionic metabolite isomers is probably due to aspatially-close structures that resembles those of the neutral, activeanticholinergics. In the active 2R isomers, while 2R1′R3′R-GA showed alower value (6.32), all others showed a similar moderate contractionactivity (about 7.15 to 7.45).

Mydriatic Activities

The mydriatic effects of the fully resolved eight zwitterionic isomerswere compared to those of (±)GA, 2R-GA, glycopyrrolate and tropicamidein vivo in rabbits. Following a 100 μl topical administration, themydriatic responses were recorded at appropriate time-intervals as %changes in pupil size. The maximum response (R_(max), % change in pupilsize at 30 min to 1 h after administration) and area under theresponse-time curve (AUC^(eff) _(o-168h)) are shown in Table 6.

TABLE 6 Maximum response (R_(max), maximum % change in pupil size) andarea under the response-time curve (AUC_(eff)) after topicaladministration (0.1 mL).^(a) Compound Conc. (%) R_(max) (%) AUC^(eff)_(0-168 h) (±)GA^(b) 0.01 1.85 ± 2.14 0.7 ± 0.9 1 45.37 ± 8.19  119 ±34  2R-GA^(b) 0.01 31.00 ± 7.14  73 ± 24 0.1 50.34 ± 7.92  182 ± 40 2R1′R3′R-GA (8a) 0.1 24.40 ± 8.33  89 ± 50 2R1′S3′R-GA (8b) 0.1 51.79 ±16.62 308 ± 106 2R1′R3′S-GA (9a) 0.1 43.90 ± 7.63  216 ± 29  2R1′S3′S-GA(9b) 0.1 47.32 ± 19.64 274 ± 134 2S1′R3′R-GA (18a) 0.1 0.00 ± 0.00 0 ± 00.4 7.44 ± 0.60 11 ± 1  2S1′S3′R-GA 18(b) 0.1 3.87 ± 4.49 13 ± 15 0.414.88 ± 1.19  37 ± 3  2S1′R3′S-GA 19(a) 0.1 0.00 ± 0.00 0 ± 0 0.4 0.00 ±0.00 0 ± 0 2S1′S3′S-GA 19(b) 0.1 3.87 ± 4.49 13 ± 15 0.4 11.01 ± 3.81 28 ± 2  glycopyrrolate^(b) 0.05 48.73 ± 12.66 2476 ± 847  0.1 52.95 ±10.93 3732 ± 866  tropicamide^(b) 0.5 44.64 ± 11.17 451 ± 121 ^(a)Datarepresent mean ± SD of four trials. ^(b)Data adapted from other testing.

The results indicate that, as in the in vitro studies, the 2R isomersare much more potent than the 2S isomers (even when the 2S dose wasincreased to 0.4%); and 2R1′R3′R-GA is less potent than the other three2R isomers. In FIG. 4, the activity-time profiles of four 2R and one 1′Sisomers (the most active S isomer) at 0.1% are displayed. Thepupil-dilating potency of the most potent three 2R isomers at a dose of0.1% is similar to that of 0.05 to 0.1% of glycopyrrolate and 0.5% oftropicamide, however, their duration of actions was much shorter thanthat of the “hard” glycopyrrolate (AUC 200-300 vs. 2500, respectively),and somewhat shorter than that of tropicamide, in agreement with softdrug design principles. The activities of 2R1′S3′R-GA (the most activezwitterionic isomer) and glycopyrrolate lasted for 10 h and 144 h,respectively, as displayed in FIG. 5. These results indicate that a goodpharmacological effect can be achieved by some 2R zwitterionic isomers,and these isomers can be rapidly eliminated from the body. Furthermore,the active 2R zwitterionic isomers did not cause any observableirritation reactions, such as eye-closing, lacrimation, mucous dischargeas well as change in the intraocular pressure during the topicalapplications; and unlike other conventional anticholinergics, these 2Rzwitterionic isomers did not induce dilation of the pupil in thecontralateral (water-treated) eyes, indicating no or low systemicside-effects. Therefore, these soft drugs are safe, promising shortacting anticholinergics with the possibility of largely reduced unwantedside effects.

Conclusion

Isomers of N-substituted soft anticholinergics based on glycopyrrolate,the methyl and ethyl esters, and their zwitterionic metabolite weresynthesized and separated. Their pharmacological activities wereevaluated in vitro and in vivo. The receptor binding (pK_(i)) resultsindicate that stereo-specificity and stereo-selectivity are veryimportant in these soft anticholinergics. There were three chiralcenters presented in the structure of these compounds. The mostsignificant improvement of the receptor binding activity was observed in2R configuration, followed by 1′S. The activities of 3′R and 3′S couldbe affected by the configurations of the other two chiral centers. Theimprovement of M₃1M₂ muscarinic-receptor subtype-selectivity was foundmost significant in 2R3′S configurations followed by 2R3R. Theconfiguration of chiral center 1′ showed no effect on M₃/M₂muscarinic-receptor subtype-selectivity. Comparable results obtainedfrom guinea pig ileum assays (pA₂), and rabbit mydriasis test onzwitterionic isomers further confirmed the stereo-specificity of theseanticholinergics. The pharmacological potency of eight zwitterionicisomers was determined to be2R1′S3′S=2R1′S3′R=2R1′R3′S>2R1′R3′R>2S1′S3′R>2S1′S3′S=2S1′R3′R>2S1′R3′S(student t-test, p<0.05). When topically administered (0.1%) in rabbiteyes, some 2R-zwitterion isomers (2R1′S3′S, 2R1′S3′R and 2R1′R3′S)showed similar mydriatic potencies to that of glycopyrrolate andtropicamide, however, their mydriatic effects were of considerablyshorter duration, and they did not induce dilation of the pupil in thecontralateral, water-treated eyes, indicating that, in agreement withtheir soft nature, they are locally active, but safe and have a lowpotential to cause systemic side effects. The usefulness and safety ofthese glycopyrrolate-based soft anticholinergics have been thereforefurther proved.

Further Synthesis and Biological Testing

A series of pure stereoisomeric soft glycopyrrolate analogues 3, 4 and 5below was synthesized by using chiral intermediates and by carefulseparation of the stereoisomers formed during the last quaternizationstep of the synthesis. The stereochemistry of the products waselucidated by using various 1D and 2D NMR techniques. Anticholinergicactivity of the new compounds was determined by receptor binding studiesand further by performing tests on isolated organs and by in vivo tests.Receptor binding revealed that in the higher alkyl ester series the(2R,1′R,3′R) and the (2R,1′S,3′S) isomers were the compounds showing thehighest receptor affinity; furthermore, it demonstrated the confines ofthe length of the alkyl chain. In vitro isolated organ experimentscorrelated well with the receptor binding results, and in vivoinvestigations indicated the soft character of the compounds.

One of the most effective anticholinergic compounds is glycopyrrolate[3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1,1-dimethylpyrrolidiniumbromide, 1 below] containing a quaternary N-atom the charge of whichprevents its crossing through lipid membranes and therefore, compared toe.g atropine, glycopyrrolate has reduced CNS-related side effects. Themolecule contains two chiral centers: at position 2 of the acyl groupand at position 3′ of the pyrrolidinyloxy moiety and hence this compoundcan exist in the form of the four stereoisomers (2R,3′R), (2R,3′S),(2S,3′R), (2S,3′S). The marketed drug is a mixture of stereoisomerswhile the (2R,3′R) form is known as Ritropirronium bromide.

Soft analogues of glycopyrrolate such as compounds of formulas 2, 3 and6 below containing three chiral centers (in addition to the two centersin the parent molecule the quaternary nitrogen bearing two differentsubstituents is also asymmetric) have also been described above and havebeen shown to possess the expected soft character. This has beenreflected by the relatively short duration of action and low systemicside effects. In addition, it has been shown above that enzymatichydrolysis of the esters 2 and 3 yielded the corresponding zwitterionicacid 6 which is much less active in rats and is rapidly eliminated. Theinitially prepared soft analogues were either mixtures of all possibleeight stereoisomers or mixtures of four stereoisomers containing the(R)-form of the cyclopentylmandeloyl unit while the remaining two chiralcenters were in racemic forms.

Soft analogues of Glycopyrrolate

R 2 methyl 3 ethyl 4 n-hexyl 5 n-octyl 6 H

Considering the favorable biological test results of compounds 2 and 3the question emerged whether any of the pure stereoisomers could haveany advantage over the others or not. Therefore, the aim of the presentwork was to synthesize and test pure stereoisomers of the softglycopyrrolate analogues 3, 4 and 5 and at the same time to study theinfluence of higher alkyl groups as R upon the extent and time course ofanticholinergic activity. Thus, the primary target molecules were thepure stereoisomers of the hexyl esters 4 and octyl esters 5 with theproviso that the configuration of C-2 in the cyclopentylmandeloyl unitwas fixed as (R) since literature data showed (R)-cyclopentylmandeloylderivatives to be more active anticholinergics than their(S)-counterparts. This meant that only two chiral centers (N-1′ and C-3′in the pyrrolidinyloxy moiety) and consequently only four stereoisomershad to be taken into consideration. In addition, for comparison,analogous pure stereoisomers of the ethyl esters 3 were alsosynthesized,

Synthesis of the Pure Stereoisomers

The target compounds were prepared by quaternization of the keyintermediates 10 (see Scheme 4) with the bromoacetates 11, 12 and 13,respectively, wherein 10 was used either as a mixture of the (2R,3′S)and (2R,3′R) diastereomers (Method A) or as the individual diastereomers(Method B).

The diastereomeric mixture of compound 10 was prepared first by theknown transesterification of (R)-methyl cyclopentylmandelate (8) withracemic 1-methyl-3-pyrrolidinol [(R,S)-(9)]. Later it was found thatmuch higher yield could be reached by direct coupling of(R)-cyclopentylmandelic acid (7) with (R,S)-(9) under Mitsunobuconditions. On the other hand, the individual diastereomers of 10 wereobtained via two different routes. Thus, transesterification of(R)-methyl cyclopentylmandelate (8) with (S)-1-methyl-3-pyrrolidinol[(S)-(9)] as above proceeded with retention of configuration at C-3′ andyielded (2R,3′S)-10, On the other hand, direct coupling of(R)-cyclopentylmandelic acid (7) with the same (S)-(9) under Mitsunobuconditions (with inversion of configuration at C-3′) led to (2R,3′R)-10.

Next, in Method A, quaternization of the mixture of (2R,3′S)-10 and(2R,3′R)-10 with the bromoacetates 12 and 13, respectively, giving riseto the formation of a new chiral center at N-1′, led to mixtures of thefour stereoisomeric target compounds 4a-d and 5a-d, respectively. Theindividual stereoisomers could be separated only partially by acombination of various chromatographic and crystallization methods (seeExperimental below).

Isolation of the pure stereoisomers was simpler in the other approach:in Method B, quaternization of (2R,3′S)-10 with the bromoacetates 11 and12, respectively, as above afforded only two stereoisomers, i.e. the(2R,1′R,3′S) and the (2R,1′S,3′S) versions both of compounds 3 and 4,respectively, and separation of these components was achieved with lessdifficulty. On the other hand, in full analogy with the abovesaid, thefinal quaternization starting with (2R,3′R)-10 resulted in the formationof the other pair of isomers, i.e. the (2R,1′R,3′R) and the (2R,1′S,3′R)versions of the final products 3 and 4, respectively. Finally, fromamong the twelve target compounds (3a-d, 4a-d and 5a-d) two pairs ofcompounds, i.e. 3a+3b and also further 5a+5d were obtained asinseparable mixtures while all other stereoisomers could be isolated inpure state. Note that 3a-d are also referred to herein as Compounds (k),(l), (m) and (n), respectively; 4a-d are also referred to herein asCompounds (o), (p), (q) and (r), respectively; and 5a-d are alsoreferred to herein as Compounds (s), (t), (u) and (v), respectively.

Structure Elucidation and Assignment of Stereochemistry

The structures and stereochemistry of the new target compounds wereelucidated by detailed NMR studies and the purity of the samples wasconfirmed by HPLC. The assignment of the individual stereoisomers isillustrated below using the example of the four isomeric hexyl esters4a-d. A complete ¹H and ¹³C NMR signal assignment was achieved byapplying ¹H, ¹³C, DEPT, and two-dimensional ¹H—COSY, ¹H, ¹H-TOCSY and¹H, ¹³C—HSQC correlation experiments. The characteristic ¹H and ¹³Cchemicals shifts are compiled in Table 7. Due to the high similarity ofthe chemical shifts of the isomers 4a-d, a simple differentation of thestructures was not possible, only the ⁺NCH₃ and ⁺NCH₂COOR chemicalshifts exhibited characteristic differences. To reveal thestereochemistry, one-dimensional selective NOESY, two-dimensional ROESYand NOESY spectra were run, affording evidences of interprotonicdistances less than 5 Å. The double arrows in Scheme 5 denote thedetected relevant NOE ¹H/¹H steric proximities.

TABLE 7 Characteristic ¹H and ¹³C chemical shifts of isomers 4a-d inCDCl₃. 4a [(o)] 4b [(p)] 4c [(q)] 4d [(r)] 2R, 1′R, 3′S 2R, 1′S, 3′S 2R,1′R, 3′R 2R, 1′S, 3′R ¹H ¹³C ¹H ¹³C ¹H ¹³C ¹H ¹³C 1 — 174.5 — 174.6 —174.6 — 174.5 2 — 79.8 — 79.7 — 79.4 — 79.8 2′_(cis) 4.36 70.3 4.16 68.93.91 70.0 4.20 70.5 2′_(trans) 4.46 4.68 4.55 4.38 3′ 5.52 73.1 5.5773.4 5.55 73.3 5.53 73.1 4′_(cis) 2.06 31.5 1.96 30.1 2.24 29.6 2.2530.1 4′_(trans) 2.79 2.93 3.05 2.88 5′_(cis) 4.08 65.0 4.03 65.1 4.1765.1 4.20 65.3 5′_(trans) 4.18 4.39 4.44 4.34 NCH_(3 cis) — 3.24 50.73.03 50.6 — NCH_(3 trans) 3.69 51.9 — — 3.69 51.9 NCH_(2 cis) 4.74; 62.8— — 4.52; 62.9 4.86 4.69 NCH_(2 trans) — 5.16; 63.8 5.08; 63.7 — 5.205.19 Ph_(ipso) 141.4 141.1 140.8 141.2 Ph_(ortho) 7.59 126.1 7.57 126.07.58 126.0 7.59 126.0 Ph_(meta) 7.34 128.5 7.36 128.6 7.37 128.6 7.36128.6 Ph_(para) 7.27 128.2 7.30 128.2 7.27 128.0 7.27 128.0 HC—C-2 2.8746.8 2.96 46.1 2.93 45.6 2.89 46.8

Selective irradiation of the ⁺NCH₃ signal in 4b resulted in NOEintensity enhancement at the and at the ortho hydrogen signals, whichunambiguously proved the 1′S configuration and at the same time, thedepicted preferred conformation of the 0-acyl moiety. Irradiation of theNCH₃ signal in 4a marked out only the hydrogen atom H_(trans)-2′,located on the same side of the pyrrolidine ring. In the case ofcompound 4d, the appearance of a strong ⁺NCH₃/H-3′ cross peak in theROESY spectrum gave evidence of the 1′S configuration, whereas theH-3′/H_(ortho) response revealed the conformation of the O-acyl group.

In compound 4c due to the unfavourable signal overlapping (e.g. ⁺NCH₃and H-4′_(trans)), the two-dimensional measurement does not work. Here,the one-dimensional selective NOESY was utilized again. Irradiating theH_(ortho) hydrogen atoms a small, but significant NOE was observed atthe ⁺NCH₃ signal, which is in accordance with the depicted configurationand conformation.

Due to the pseudorotation of the pyrrolidine ring and the highflexibility of the compounds 4a-d, conformational averaged structuresare expected. Despite this, the anomalous upheld shift of the ⁺NH₃signals (3.24 and 3.03 ppm) can be explained by the well knownanisotropic shielding effect of the aromatic ring, wherein the hydrogenatoms located above the plane of the aromatic ring show smaller chemicalshifts. The smaller chemical shifts of the NCH₂ (cis) hydrogens in 4a(4.74; 4.86) and 4d (4.52; 4.69) are in accord with the relative stericarrangement. Preference of the conformations of compounds 4a-d, wherethe aromatic ring is oriented towards the nitrogen atom, is in accordwith the stabilization of the positive charge on the nitrogen by then-system of the phenyl group.

Biology: Evaluation of the Anticholinergic Activity

Receptor Binding

Evaluation of the affinity of the soft glycopyrrolate analogues 3a-d,4a-d and 5a-d above for muscarinic receptors was carried out using[³H]QNB as ligand and rat cortical membrane preparation as a source ofthe receptor. The affinity (summarized in Table 8) of these compoundsfor the muscarinic receptors (mainly M₁ in this preparation) was foundone or two orders of magnitude lower than those of the referencecompounds glycopyrrolate (Ki=0.8 nM) and atropine (Ki=1.9 nM) but theinstant compounds were still strong antagonists of the muscarinicreceptor, The nature of the interaction was characterized by the steepHill slope, the value of which was close to unity indicating theantagonistic action. The difference between the effects of the purestereoisomers was seen most clearly within the hexyl series as in thiscase all the four possible stereoisomers were isolated in pure state.The compounds 4b (2R,1′S,3′S) and 4c (2R,1′R,3′R) were equally,approximately four-fold, more active than 4a (2R,1′R,3′S) or 4d(2R,1′S,3′R). The same tendency was clear in case of the less activeoctyl series (5b, 5c), even though the other two isomers (5a+5d) weretested as a mixture. The above (2R,1′S,3′S) and (2R,1′R,3′R) compoundscontain the larger quaternizing group (CH₂COOR) in trans position to thecyclopentylmandeloyloxy moiety and this fact suggests that thesterically less crowded nature of these isomers may contribute to thehigher receptor affinity, in contrast with the sterically more crowdedcis isomers.

TABLE 8 Receptor binding strength of the glycopyrrolate analoguesCompound Ki (nM), Average ± SD Hill slope 3a + 3b [(k) + (l)] 65 ± 8−0.96 ± 0.09 3c [(m)] 16 ± 1 −0.93 ± 0.08 3d [(n)] 16 ± 1 −1.07 ± 0.024a [(o)] 67 ± 9 −1.22 ± 0.08 4b [(p)] 13 ± 1 −1.19 ± 0.05 4c [(q)] 15 ±1 −1.24 ± 0.04 4d [(r)] 58 ± 5 −1.15 ± 0.05 5a + 5d [(s) + (v)] 303 ± 7 −1.20 ± 0.08 5b [(t)] 60 ± 5 −1.22 ± 0.11 5c [(u)] 68 ± 6 −1.26 ± 0.06

On the other hand, in the ethyl ester series the isomers 3c (2R,1′R,3′R)and 3d (2R,1′S,3′R), i.e. the compounds wherein thecyclopentylmandeloyloxy moiety is attached to the pyrrolidine ring inthe α-position, have higher affinity indicating that in this case thesteric position of the smaller CH₂COOEt group has less influence uponreceptor affinity.

The effect of the length of the alkyl chain in the ester group upon thereceptor binding seemed to be negligible up to 6 carbon moiety but whenthe longer chain was used this already affected receptor binding(compare the whole hexyl and octyl series).

Ex Vivo Experiments with Isolated Organs

Determination of the pA₂ values in guinea pig trachea and ileum assayresulted in the expected results. In line with the receptor bindingexperiments in both of the isolated organ preparations, atropine andglycopyrrolate were more active (Table 9) than the instant selectedglycopyrrolate analogues chosen to represent compounds with markedlydifferent receptor affinities. The ethyl and hexyl side chain containingcompounds (3c and 4b) were practically equally effective while the octylchain containing esters showed weaker activity.

TABLE 9 pA₂ values in two types of isolated organ experiments* TracheaIleum Antagonist pA₂ Slope ± S.E. pA₂ Slope ± S.E. Atropine 8.85 0.98 ±0.02^(a) 8.52 0.96 ± 0.30^(a) Glycopyrrolate 9.43 1.53 ± 0.07^(a) 9.661.03 ± 0.37^(a) 3c [(m)] 8.12 1.54 ± 0.18^(a) 8.48 0.76 ± 0.08^(a) 4b[(p)] 8.21 1.14 ± 0.10^(a) 8.14 1.31 ± 0.67^(a) 5a + 5d [(s) + (v)] 7.230.79 ± 0.07  6.64 1.03 ± 0.37^(a) *data are presented of mean estimatesin tissue samples from four animals ^(a)deviation from unity is notsignificant (P > 0.05)In Vivo ExperimentsCarbachol Induced Bradycardia in the Rat

The bradycardia protective effect of the selected new compounds wascomparable both to their receptor binding affinity and their activity inthe isolated organ experiments, In line with this, their in vivoactivity (FIG. 6) was lower than that of glycopyrrolate (GP) but moreimportantly their duration of action was notably shorter than that ofthe parent compound, indicating their potential soft character.

Experimental

Chemistry

Melting points were determined on a Boetius microscope and areuncorrected. Purity of the compounds was tested on TLC plates (silicagel, Merck). The spots were visualized under UV light and/or by exposureto iodine vapours. NMR spectra were recorded in CDCl₃, DMSO-d₆ or CD₃ODsolutions using a Bruker Avarice 500 spectrometer, operating at 500/125MHz (¹H/¹³C). Chemical shifts are given on the δ-scale and werereferenced to TMS. Pulse programs for the 1D and 2D NMR experiments weretaken from the Bruker software library. For structure elucidation andNMR signal assignment ¹H, ¹³C, DEPT-135, selective 1D-NOESY, ¹H,¹H—COSY,¹H,¹H-TOCSY, ¹H,¹³C—HSQC, ¹H,¹³C-¹HMBC, ¹H,¹H—ROESY and ¹H,¹H—NOESYspectra were recorded.

Analytical HPLC of compounds 3, 4 and 5 was performed using a Waters(Milford, Mass.) HPLC system consisting of a model 510 isocratic pumpworking at 1 ml/min flow rate, a WISP programmable autoinjector with 10μl injection volume and a model 486 single channel variable wavelengthUV detector with 220 nm preset wavelength. The applied HPLC stationaryphase was a Prontosil 120 C18 AQ 5 μm column with 150*4 mm geometry.Column temperature: 40° C. The optimal mobile phase was a mixture of 30mM ammonium acetate/MeOH/acetonitrile, in a ratio of 55/17.5/27.5(v/v/v) for compounds 3, in a ratio of 34/16/55 (v/v/v) for compounds 4and in a ratio of 18/20/60 (v/v/v) for compounds 5. The enantiomericpurity of 2-cyclopentylmandelic acid (7) was determined by chiral ligandexchange chromatography on a Nucleosil Chiral-15 μM, 250*4 mm chiralHPLC column. The mobile phase was 0.5 mM CuSO₄/acetonitrile 97/3 (v/v),flow rate: 1 nil/min, column temperature: 60° C., detection wavelength:220 nm. The retention time of the individual enantiomers was 13.5 min(R) and 15.5 min (S), respectively. The observed selectivity was 1.18.

(R)-2-Cyclopentylmandelic acid [(R)-(7)] was obtained by resolution ofthe racemic acid with (−)-cinchonidine, (R)-methyl2-cyclopentylmandelate [(R)-(8)] was prepared as described in the artwhile (R,S)- and (S)-1-methyl-3-pyrrolidinol, respectively [(R,S)- and(S)-(9), respectively], were synthesized in two steps starting with(R,S)- and (S)-malic acid, respectively, as previously described. Ethylbromoacetate (11) was purchased from Aldrich while the homologousn-hexyl (12) and n-octyl bromoacetates (13) were prepared by reaction ofthe corresponding alcohol with bromoacetyl bromide. Found values ofelemental analyses agreed with calculated values within the range of±1%.

Preparation of the Quaternized Target Compounds 3, 4 and 5

Method A

A mixture of (2R,3′R)-10 and (2R,3′S)-10 (1.0 mM), together with thealkylating agent 12 or 13 (2.0 mM) in acetonitrile (12 ml) was stirredfor 2 hours at room temperature. After completion of the reaction, thesolvent was evaporated and the products were isolated as given below inthe description of the individual compounds.

Method B

A mixture of (2R,3′R)-10 or (2R,3′S)-10 (0.3 mM) and the alkylatingagent 11 or 12 (0.6 mM) in acetonitrile (5 ml) was allowed to react andthe crude product was isolated as described under Method A above.

(2R,1′R,3′S)3-(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide (3a) [Compound (k)] and (2R,1′S,3′S)3(2-cyclopentyl-2-phenyl-2-hydroxyacetoxyl)-1-ethoxycarbonyloxymethyl)-1-methylpyrrolidiniumbromide (3b) [Compound (l)]

Method B above was followed starting with (2R,3′S)-10. The crude productwas purified by silica gel chromatography eluting withchloroform-methanol 9:1 and 3a and 3b were isolated as an inseparablemixture. Yield: 54%, mp. 163° C., ratio 3a/3b (¹H-NMR): 4:1.

3a NMR (CDCl₃) δ 3.68 (3H, s, NCH₃), 4.78 d, NCH₂), 4.89 (1H, d, NCH₂),5.55 (1H, m, H-3); 3b ¹H NMR (CDCl₃) δ 3.27 (3H, s, NCH₃), 5.26 (1H, d,NCH₂), 5.30 (1H, d, NCH₂), 5.51 (1H, m, H-3).

(2R,1′R,3′R)3-(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide (3c) [Compound (m)] and (2R,1′S,3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl-methylpyrrolidiniumbromide (3d) [Compound (n)]

Method B above was followed starting with (2R,3′R)-10 and the products3c and 3d were separated by silica gel chromatography of the crudeproduct eluting with chloroform-methanol 9:1.

3c: yield: 19%, mp. 98° C., purity (HPLC): 93%.

¹H NMR (CDCl₃) δ 1.29 (3H, t, CH ₃CH₂O), 2.24 (1H, m, H_(c)-4′), 2.94(1H, m, HC—C-2), 3.02 (3H, s, NCH₃), 3.07 (1H, m, H_(t)-4′), 3.88 (1H,m, H_(c)-2′), 4.13 (1H, m, H_(c)-5′), 4.23 (2H, q, CH₃ CH ₂O), 4.46 (1H,m, H_(t)-5′), 4.56 (1H, m, H_(t)-2′), 5.10 (1H, d, NCH₂), 5.22 (1H, d,NCH₂), 5.56 (1H, m, H-3′), 7.28 (1H, t, Ph_(p)), 7.37 (2H, t, Ph_(m)),7.58 (2H, d, Ph_(o)).

3d: yield: 28%, mp. 70° C., purity (HPLC): 96%.

¹H NMR (CDCl₃) δ 1.33 (3H, t, CH ₃CH₂O), 2.24 (1H, m, H_(c)-4′), 3.66(3H, s, NCH₃), 4.65 (1H, d, NCH₂), 4.74 (1H, d, NCH₂), 5.54 (1H, m,H-3′), 7.27 (1H, t, Ph_(p)), 7.35 (2H, t, Ph_(m)), 7.59 (2H, d, Ph_(c)).

(2R,1′R,3′S)3(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(n-hexyloxycarbonylmethyl)-1-methylpyrrolidiniumbromide (4a) [Compound (o)] and (2R,1′S,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(n-hexyloxycarbonylmethyl)-1-methylpyrrolidiniumbromide (4b) [Compound (p)]

Method B above was followed starting with (2R,3′S)-10 and the products4a and 4b were separated by silica gel chromatography of the crudeproduct eluting with chloroform-methanol 9:1.

4a: yield: 18%, mp. 146° C., purity (HPLC): 93%.

4b: yield: 23%, mp. 125-128° C. purity (HPLC): 96%.

For NMR data of 4a-b see above.

(2R,1′R,3′R)3-(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(n-hexyloxycarbonylmethyl)-1-methylpyrrolidiniumbromide (4c) [Compound (q)] and (2R,1′S,3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy-1-(n-hexyloxycarbonylmethyl)-1-methylpyrrolidiniumbromide (4d) [Compound (r)]

Method B above was followed starting with (2R,3′R)-10 and the products4c and 4d were separated by silica gel chromatography of the crudeproduct eluting with chloroform-methanol 9:1.

4c: yield: 20%, mp, 138° C., purity (HPLC): 98%.

4d: yield: 55%, mp. 116° C., purity (HPLC): 95%.

For NMR data of 4c-d see above.

As an alternative, upon preparing compounds 4a-d by following Method Aabove, the products 4 h and 4c could be isolated in pure state asdescribed below while 4a and 4d were obtained in the form of aninseparable mixture. Thus, the crude product was triturated with ethylacetate to give the mixture 4a+4d [Compounds (o) and (r)]; as a solid,yield: 42%. The mother liquor was concentrated to dryness and theresidue was purified by column chromatography on silica gel eluting withchloroform-methanol 9:1. Subsequently compounds 4b (yield: 12%)[Compound (p)]; and 4c (yield: 6%) [Compound (q)] were separated bypreparative thin layer chromatography developing withchloroform-methanol 9:1.

(2R,1′R,3′S)3-(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(n-octyloxycarbonylmethyl)-1-methylpyrrolidiniumbromide (5a) [Compound (s)], (2R,1′S,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(n-octyloxycarbonylmethyl)-1-methylpyrrolidiniumbromide (5b) [Compound (t)], (2R, 1′R,3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(n-octyloxycarbonylmethyl)-1-methylpyrrolidiniumbromide (5c) [Compound (u)] and (2R,1′S,3′R),3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(n-octyloxycarbonylmethyl)-1-methylpyrrolidiniumbromide (5d) [Compound (v)]

By following Method A above and purification of the crude product bysilica gel column chromatography eluting with chloroform-methanol 9:1the compounds 5a and 5d [Compound (s) and (v)] were obtained in the formof a mixture inseparable by TLC and HPLC, On the other hand, 5b[Compound (t)] and 5c [Compound (u)] could be separated by a finalpreparative thin layer chromatography, developing withchloroform-methanol 9:1.

Mixture of 5a and 5d: yield: 47%, ratio 5a/5d (¹H-NMR): 1:1.

5a ¹H NMR (CDCl₃) δ 2.10 (1H, m, H_(c)-4′), 3.64 or 3.67 (3H, s, NCH₃),4.71 (1H, d, NCH₂), 4.81 (1H, d, NCH₂); 5d ¹H NMR (CDCl₃) δ 2.28 (1H, m,H_(c)-4′) 3.64 or 3.67 (3H, s, NCH₃), 4.54 (1H, d, NCH₂), 4.67 (1H, d,NCH₂).

5b: yield: 10%, mp. 30° C., purity (HPLC): 86%.

¹H NMR (CDCl₃) δ 0.90 (3H, t, CH ₃CH₂), 2.00 (1H, m, H_(c)-4′), 2.93(1H, m, HC—C-2 and 1H, m, H_(t)-4′), 3.27 (3H, s, NCH₃), 4.10 (1H, m,H_(c)-5′), 4.18 (2H, t, CH₃ CH ₂), 4.18 (1H, m, H_(c)-2′), 4.29 (1H, m,H_(t)-5′), 4.58 (1H, m, H_(t)-2′), 5.16 (2H, s, br, NCH₂), 5.56 (1H, m,H-3′), 7.27 (1H, t, Ph_(p)), 7.35 (2H, t, Ph_(m)), 7.57 (2H, d, Ph_(o)).

5c: yield: 7.5%, purity (HPLC): 93%.

5c ¹H NMR (CDCl₃) δ 0.89 (3H, t, CH ₃CH₂), 2.28 (1H, m, 1H_(c)-4′), 2.93(1H, m, HC—C-2), 2.53 (3H, s, NCH₃), 3.05 (1H, m, 3.86 (1H, m,H_(c)-2′), 4.12 (1H, m, H_(c)-5′) 4.16 (2H, t, CH₃ CH ₂), 4.30 (1H, m,H_(t)-5′), 4.50 (1H, m, H_(t)-2′), 4.97 (1H, d, NCH₂), 5.10 (1H, d,NCH₂), 5.58 (1H, m, H-3′), 7.27 (1H, t, Ph_(m)), 7.38 (2H, t, Ph_(m)),7.59 (2H, d, Ph_(o)).

Mixture of (2R,3′R) and (2R,3′S)3-(2-Cyclopentyl-2-phenyl-2-hydroxracetoxy)-1-methylpyrrolidine[(2R,3′R)-10 and (2R,3′S)-10]

A solution of diisopropyl azodicarboxylate (1.5 mM) in tetrahydrofuran(1 ml) was added dropwise to a mixture of (R)-7 (1.5 mM), (R,S)-9 (1.64mM) and triphenylphosphine (1.5 mM) in tetrahydrofuran (4 ml) at roomtemperature. The reaction mixture was stirred at room temperature for 2hours and the solvent was removed in vacuo. The residue was suspended inethyl acetate and extracted with 1N hydrochloric acid. The aqueoussolution was made alkaline with 5N aqueous sodium hydroxide, followed byextraction with ether. The organic layer was dried over magnesiumsulfate and concentrated in vacuo giving the title compound as acolorless oil, Yield: 87%, purity (HPLC): 97% (total area of twounresolved peaks).

1:1 mixture of (2R,3′R)-10 and (2R,3′S)-10:

¹H NMR (CDCl₃) δ 2.35 and 2.39 (3H, s, NCH₃), 2.56 and 2.68 (1H, m,H_(c)-2′), 2.94 (1H, m, HC—C-2), 3.75 (1H, s, HO—C-2), 5.24 (1H, m,11-3), 7.27 (1H, t, Ph_(p)), 7.36 (2H, t, Ph_(m)), 7.67 (2H, d, Ph_(o)).

(2R,3′S) 3-(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-methylpyrrolidine

The methyl ester (R)-(8) was submitted to transesterification with (S)-9by following the known method of Franko and Lunsford. Yield: 28%, purity(HPLC): 97%.

¹H NMR (CDCl₃) δ 2.39 (3H, s, NCH₃), 2.68 (1H, m, H_(c)-2′), 2.94 (1H,m, HC—C-2), 3.79 (1H, s, br, HO—C-2), 5.24 (1H, m, H-3′), 7.27 (1H, t,Ph_(p)), 7.35 (2H, t, Ph_(m)), 7.67 (2H, d, Ph_(o)).

(2R,3′R) 3-(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-methylpyrrolidine(2R,3′R)-10

(R)-(7) was coupled with (S)-9 under Mitsunobu conditions as describedabove under 3.1.1.7. Yield: 49%, purity (HPLC): 95%.

¹H NMR (CDCl₃) δ 2.34 (3H, s, NCH₃), 2.93 (1H, m, HC—C-2), 3.78 (1H, m,HO—C-2), 5.23 (1H, m, H-3′), 7.27 (1H, t, Ph_(p)), 7.34 (2H, t, Ph_(m)),7.67 (2H, d, Ph_(o)).

Biology: Test Methods

Receptor Binding Assay

The binding of [³H]quinuclidinyl benzylate ([³H]QNB; Amersham, 42.0Ci/mmol) to muscarinic receptors was measured according to the method ofYamamura and Snyder (1974), with some modifications, as describedpreviously by Barlocco et al. (1997). Briefly, male Sprague-Dawley rats(180-220 g) were decapitated and cerebral cortices removed, discardingthe white matter. Pooled tissue was homogenized in 10 volumes ofice-cold 50 mM Tris-HCl buffer (pH 7.4) by using a motor driven glasshomogenizer. The homogenate was centrifuged at 4° C. and 30,000 g for 10mm. The pellet was washed twice with the same butler by resuspension,followed by centrifugation at 4° C. and 30,000 g for 10 min. The finalpellet was resuspended in 10 volumes of Tris-HCl buffer and stored at−20° C. before use. Protein content was determined by the method ofBradford (1976) using bovine serum albumin as the standard.

In all binding experiments, membranes (0.25 mg/ml protein), radioligandand competing drugs were incubated in a final volume of 1 ml of 50 mMTris-HCl buffer (pH 7.4) for 60 min at 25° C. For saturation studies,membranes were incubated with 0.01-2 nM [³H]QNB. In competitionexperiments, the final concentration of the radioligand was 0.2 nM andcompeting drugs were given in S concentrations. Non-specific binding wasdetermined with 1 μM atropine. The incubation was terminated by rapidvacuum filtration over Whatman GF/B filters using a Brandel CellHarvester. Samples were washed immediately with 3×4 ml ice-cold Tris-HClbuffer and placed in 6 ml scintillation fluid. Radioactivity wasestimated by liquid scintillation counting.

Data are the mean±S.E.M. of at least three experiments run in duplicate.GraphPad Prism 3.0 (GraphPad Software Inc., San Diego, Calif., USA) wasused to perform linear and non-linear regression analysis of the data.Saturation binding parameters (K_(d) and B_(max)) were determined bylinear regression analysis of the transformed saturation binding data.Competition binding isotherms were analyzed by non-linear regression toderive estimates of the IC₅₀ values and Hills slopes. IC₅₀ values wereconverted to K_(i) values according to the equation of Cheng and Prusoff(1973).

Guinea Pig Trachea and Ileum Assay (pA₂ Value Determination)

Tracheal preparations were made as described previously in details byPreuss and Goldie (1999). Briefly: tracheas were isolated from maleDunkin-Hartley guinea pigs (280-300 g) and the ring preparations (2-3 mmin width) were suspended under 500 me resting tension in an organ bathcontaining Krebs' bicarbonate buffer aerated with 95% O₂/5% CO₂. Changesin isometric tension were measured by a force displacement transducer(Experimetria, Budapest, Hungary) coupled to a Watanabe recorder.Cumulative concentration-effect curves were constructed to carbachol inthe absence or presence of the antagonists. In each animal twopreparations were used as time control (i.e., repeated carbachol curvesin the absence of any antagonist) the remaining two preparations wereused to test responses in the presence of two different concentrationsof the antagonist.

Antagonists were added to the organ bath 30 min prior to commencement ofthe agonist concentration versus effect curves. Schild plots wereconstructed for the antagonists against carbachol and pA₂ values as wellas slope estimates were obtained.

The ileum longitudinal muscle strips with adhering myenteric plexus werealso prepared from male guinea-pigs (200-400 g). A segment of smallintestine (8-10 cm) 10 cm proximal to the ileo-coecal valve wasdissected. The longitudinal muscle strip was obtained by mountingsegments of the whole ilea on a 1 ml pipette and gently tearing away theouter longitudinal muscle layer with a cotton swab. Longitudinal musclestrips were cut into 3-4 cm pieces. The strips were mounted in an organbath containing Tyrode solution at 37° C. under a resting tension of 500mg. Contractions were recorded isometrically with the same strain gaugesystem as above and registered on the Watanabe type polygraph. Thetissues were left to equilibrate for 30 min. Dose-response curves to theagonist were constructed by addition of acetylcholine in increasingconcentrations. The doses were given at 10 min intervals with 1 mincontact time. After a 40 min equilibration period, the preparations wereincubated with the antagonist for 20 min, and a secondconcentration-response curve to acetylcholine was constructed. Theagonist (Ach) was non-cumulatively added at 10 min intervals(concentration range: 10⁻¹⁰ to 10⁻⁵ M). Antagonists were applied in theconcentration range of 10⁻⁸ to 10⁻⁵ M, depending on the individual testcompound,

Responses were measured as changes in isometric tension and calculatedas a percentage of the maximum response attained in the initialconcentration-response curve. Determination of antagonist potencies wasdone by constructing Schild double logarithmic plot of log (DR-1)versus-log M concentrations of the antagonist, and the slope of the plotwas computed. If the slopes of the plots were not significantlydifferent from unity, the interaction was accepted as competitive innature, and antagonist potencies were expressed as pA₂ values(Arunlakshana & Schild, 1959). If the slopes of the plots weresignificantly different from unity, the method of Ariens & Van possum(1957) was used to determine pD′ values for characterization ofnon-competitive antagonism. Statistical significance was assessed byANOVA followed by Dunnett test.

Antagonistic Effect on Carbachol Induced Bracycardia

The experimental procedure described previously in detail by Juhasz etal. (1998) was followed. Male Sprague-Dawley rats, weighing 300-350 gwere anesthetized with sodium pentobarbital (50 mg/kg i.p.). Baselineelectrocardiography (ECG) recordings were performed after 15 minstabilization periods. Needle electrodes were inserted subcutaneouslyinto the limbs of the anesthetized rats and were joined to a Watanaberecorder. Recording of the heart rate (1/min) was taken before, duringand after the administration of any of the compounds until basic ECGparameters returned to baseline at a paper speed of 25 mm/sec. All drugswere administered by direct injection into the jugular vein.Anticholinergic drugs were administered in the approximatepharmacodynamic equivalent doses (0.2, 2.0 umol/kg) at 0 time, whilecarbachol (5-8 ug/kg) was injected at −5, 5, 10, 15, 20, 30, 45, 60 min.

Synthesis and Biological Testing of Tiotropium Derivatives

Structure of the Compounds

b) Synthesis of a New Tiotropium Analog

By following the inactive metabolite approach, a metabolically sensitiveester function was introduced into the tiotropium molecule resulting ina new soft anticholinergic analog. Intermediate XIII was prepared by theknown Grignard reaction of dimethyl oxalate with 2-thienylmagnesiumbromide (XII, see Scheme 6 below). Compound XIII was then submitted toknown transesterification with scopine (XIV) catalyzed by sodium metalgiving the corresponding ester XV. Finally, quaternization with ethylbromoacetate gave the target compound II (R=Et).

2-Thienylmagnesium bromide was prepared in the usual manner frommagnesium and 2-bromothiophene in ether. Scopine (XIV) was obtained fromscopolamine hydrobromide by treatment with sodium borohydride in ethanolin moderate yield as described in GB 1,469,781 (1974).

The above synthesis of the soil; tiotropium bromide analog leads to asingle isomer in a yield of 70%. Tlc indicated no further new componentin the reaction mixture of the final quaternisation step and it could beshown by NMR (two dimensional ROESY technique) that theethoxycarbonylmethyl group is in proximity to the oxirane ring. Thisfinding is somewhat surprising as the other isomer with N-methylpointing toward the oxirane ring would be sterically less crowded.

Stereochemistry of Compound (w) obtained by two-dimensional ROESYexperiment:

The numbers denote ¹H chemical shifts and coupling constants, the doublearrows indicate steric proximities,

Preparation of6β,7β-epoxy-3β-hydroxy-8-ethoxycarbonylmethyl-8-methyl-1αH,5αH-tropaniumbromide, di-2-thienylglycolate, Compound (w)

The scopine ester, represented by formula XV in Scheme 6 above, wasprepared as described above, or by conventional methods as described inEP418716 (equivalent to U.S. Pat. No. 5,610,163). Then the scopine ester(70 mg, 0.18 ml) is dissolved in 2 ml of acetone. Ethyl bromocetate (150microliters, 0.45 mM) is added and the mixture is allowed to react at20° C. for eight days. The solvent is evaporated in vacuo, 8 ml of wateris added and the organic material is extracted with chloroform. Thedesired quaternary salt is in the aqueous phase and is obtained bylyophilization. Yield 70 mg (70%). Melting point: 115° C. Thin layerchromotography on Al₂O₃: R_(f)=0.3(CHCl₃—CH₃OH, 4:1) (3 times 4 ml). Theproduct. Compound (w), has the structural formula II shown in Scheme 6above.

Preparation of6β,7β-epoxy-3β-hydroxy-8-methyl-8-(2,2,2-trichloroethoxycarbonylmethyl-1αH,5αH-tropanium bromide, di-2-thienylglycolate, Compound (x)

To the scopine ester XV (0.5 mM) in 3 ml of anhydrous acetonitrile, 1.5mM of trichloroethyl bromoacetate was added. The mixture was stirredunder argon for three days and the acetonitrile was removed underreduced pressure. To the oily residue, 15 ml of water was added andextracted with chloroform (3 times 5 ml). The aqueous solution waslyophilized to give the product as a white solid. Yield 257 mg (79%).Melting point 105° C., R_(f)=0.65 (CHCl₃—CH₃OH, 4:1). The product hasthe structural formula:

Preparation of6β,7β-epoxy-3β-hydroxy-8-carboxymethyl-8-methyl-1αH,5αH-tropanuim,di-2-thienylglycolate inner salt, Compound (aa)

A suspension 0.35 mM Compound (x) and Zn dust (0.6 mM) in acetic acid(1.5 ml) was stirred for 3 hours. To that mixture, water (2 ml) andchloroform (2 ml) were added and filtered. The solvents were evaporatedin vacuo, 3 ml of water was added and the solution was lyophilized. Thecrude product was dissolved in methanol (2 ml) and purified bychromatography on Sephadex The resulting oil was dissolved in methanol(2 ml) and precipitated with ethyl acetate (1 ml) to give the solidproduct, Compound (aa). Yield 67 mg (38%), melting point 158-165° C.(decomp). R_(f)=0.45 (CHCl₃—CH₃OH 4:1) on aluminum oxide. The producthas the structural formula:

Pharmacology1: Receptor Binding Assay

Evaluation of the affinity of the compound was made using [³H]QNB asligand and rat cortical membrane preparation as a source of thereceptor. The ethyl ester Compound (w) bound to the muscarinic receptors(mainly M₁ in this preparation) with high affinity (Table 10) althoughthis affinity was several-fold lower than those of the referencecompounds. The steep Hill slope close to unity indicates theantagonistic nature of its action.

TABLE 10 Affinities of tiotropium ethyl ester derivative [Compound (w)]and reference compounds for muscarinic receptors Compounds K_(i) (nM)Hill Slope Number of exps. Atropine 1.9 ± 0.2 −1.10 ± 0.04 4Glycopyrrolate  0.8 ± 0.10 −1.07 ± 0.03 4 Compound (w) 7.2 ± 0.5 −1.00 ±0.04 4 The K_(i) values are for inhibition of [³H]QNB binding to ratbrain cortex membranes. Values are the mean ± S.E.M. of at least threeexperiments run in duplicate.2: Experiments with Isolated Organ

In isolated organ experiments the measurement of antimuscarinic effectof Compound (w) was carried out using guinea pig tracheal ringpreparations, where smooth muscle contraction is mediated primarily bymuscarinic M₃ cholinoceptors although activation of M₂-receptors alsoplays a role in the developing contraction. Compound (w) showedexcellent activity in this test; it was more active than atropine andonly slightly less effective than ipratropium bromide (Table 11).

TABLE 11 Schild-plot analysis of the antagonism against carbachol inisolated tracheal rings of guinea pigs. Antagonist pA₂ Slope ± S.E.atropine 8.85 0.98 ± 0.02^(a) ipratropium Br 9.18 1.11 ± 0.14^(a)Compound (w) 8.82 1.03 ± 0.08^(a) Data are presented of mean estimatesin tissue from four animals pA₂: the abscissa intercept of theSchild-plot drawn ^(a)Indicates slope estimates not significantlydifferent (P > 0.05) from unity.3. Determination of the Antagonistic Effect of Anticholinergic Agents onCharbachol Induced Bradycardia in Anesthetized Rats

Intravenous administration of the cholinomimetic carbachol causes sinusbradycardia (increasing the PP cycle and RR cycle length of the ECG) inanesthetized rats. This effect, which is mediated mainly by muscarinicM₂ receptors, can be prevented by prior administration ofanticholinergic agent. The bradycardia protective effect of Compound (w)was compared to those of atropine and ipratropium bromide in thissystem. See FIG. 7. Compound (w) was less active than an equimolar doseof atropine, and was slightly less active than a 10-fold lower dose ofipratropium bromide indicating that Compound (w) may have lower affinityfor M₂ than M₁ or M₃ muscarinic receptors. See FIG. 7.

Examination of the Time Course of the Anticholinergic Effect of Compound(w) and Compound (aa) in Electrically Stimulated Guinea Pig Trachea

Experimental Procedures:

The procedure described by Takahashi T. et al., (Am J Respir Crit CareMed, 150:1640-1645, 1994) was used with slight modifications.

Male Dunkin-Hartley guinea pigs (300-500 g) were exterminated; thetracheas were rapidly removed, and placed in oxygenated normal Krebsbuffer solution. The epithelium was removed and the trachea was spirallycut into 15 mm long strips. Two strips from one animal were prepared andsuspended between parallel stainless steel wire field electrodes in10-ml organ baths containing buffer solution, which was continuallygassed by a 95% 0₂ and 5% CO₂ mixture. The tissues were allowed toequilibrate for 1 h with frequent washing, under a resting tension of1.0 g.

Indomethacin 10⁻⁵(M) was present throughout the studies to block theformation of endogenous prostaglandins. Before the experiment, capsaicin(10⁻⁵M) was added and washed out 30 min after the pre-treatment todeplete endogenous tachykinins. Tissues were also pretreated withpropranolol 10⁻⁶M) 10 min before the experiment to inhibit the effectsof endogenous catecholamines.

Isometric contractile responses were measured using force-displacementtransducers (Experimetria, Hungary) connected to a Watanabe polygraph. Astimulator (CRS-ST-01-04, Experimetria) provided biphasic square-waveimpulses with a supramaximal voltage of 40 V at source and 0.5 msduration. Stimulations were applied at a frequency of 4 Hz for 15 secfollowed by a 100 sec resting interval. After at least four stableresponses of equal magnitude were obtained, the antagonist (submaximaldose) was introduced and was left in the system until the maximal effectof the drug was observed. Thereafter the test drug was washed out.Further stimulations were delivered for at least 6 additional hours oruntil the responses returned to about 50% of the original responses.Appropriate time controls were run in parallel for all studies.

Statistical Analysis

Contractile responses were expressed as the percentage of the ownmaximal contraction. The time for offset t_(1/5) or t_(1/2) of actionwas defined as the time from washout of the test antagonist toattainment of 20 or 50% recovery of cholinergic responses.

Results:

Typical tracings were obtained during the experiments. Continuous,stable, long-lasting contraction is achieved with electricalstimulation. Upon the addition of anticholinergic agents the inhibitiondevelops with varying speed, and the inhibitory effect of the compoundslast for very different periods after the washout. In FIG. 8, the timecourse of action of the different anticholinergic compounds is shown;calculated results are summarized in Table 12, In FIG. 9, the timecourse of the inhibition of the examined compounds are displayed; theresults calculated from this data are summarized in Table 13.

The differences between the on and off rates of the Compounds (w) and(aa) are very notable.

TABLE 12 Time course of action of different anticholinergics inelectrically stimulated guinea pig tracheal strips Compound t_(1/5)onset (min) t_(1/2) onset (min) Atropine 2.5 4.6 Ipratropium Br 3.0 7.0Tiotropium 8.0 12 Cpd (w) 0.6 1.2 Cpd (aa) 0.9 2.0

TABLE 13 Time course of recovery from inhibition after washing out ofdifferent anticholinergics in electrically stimulated guinea pigtracheal strips Compound t_(1/5) offset (min) t_(1/2) offset (min)Atropine 10 31 Ipratropium Br 6.5 19 Tiotropium >360 >360 Cpd (w) 60 130Cpd (aa) 27 140Investigation of the Anticholinergic Action of Compounds inAcetylcholine Induced Bronchoconstriction in Anesthetized Guinea PigsExperimental Procedure

Male Hartley guinea pigs (320±120 g) (Charles River) were housed understandard conditions. Guinea pigs were anesthetized with urethane (2g/kg, intraperitoneally), the trachea was cannulated and the animal wasrespired using a small animal respiratory pump (Harvard Apparatus LTD,Kent UK). Respiratory hack pressure was measured and recorded using arodent lung function recording system (MUMED, London UK). For drugadministration the right jugular vein was cannulated. Following thesurgical preparation guinea pigs were allowed to stabilize for 20minutes. Ten minutes before acetylcholine administration the animalswere disconnected from the ventilator and either the vehicle (10 mglactose) or different amounts of the drug (suspended in the same amountof vehicle) were administered intratracheally. The trachea wasreconnected to the ventilator and changes in pulmonary mechanics werefollowed. Acetylcholine (10 μg/kg) was administered intravenously inevery 10 minutes six times.

Results

Compounds (q) and (m) of the invention and glycopyrrolate all exhibiteda protective effect on the acetylcholine-induced bronchoconstrictionprovoked in this test. Glycopyrrolate was administered at a dose of 0.01mg/kg, while Compounds (q) and (in) were administered at a dose of 0.1mg/kg. See FIG. 10.

This test is a model for asthma, chronic obstructive pulmonary disorderand other obstructive respiratory tract disorders in which theeffectiveness of the compounds of formulas (Ia) and (Ib) can beevaluated.

Test for Bronchodilatory Effect of inhaled Test Compounds in Balb/c Mice

Female BALB/c mice, weight range 19-22 g, are obtained, for example fromCharles River Laboratories (Kingston, N.C.). They receive food and waterad libitum.

Compounds for aerosol administration are prepared in sterile Dulbecco'sPhosphate Buffered Saline. Mice are placed in a carousel-style, noseonly, exposure chamber and allowed to inhale aerosols for five minutes,using an ICN SPAG-2 nebulizer. This nebulizer generates a mean aerosolparticle size of 1.3 microns at a rate of approximately 0.25 ml/minute.

Ten minutes and 36 hours later, the mice are moved to whole bodyplethysmograph chambers. Bronchoconstriction is induced in the mice byadministration of an 80 mg/ml methacholine (MC) aerosol into theplethysmograph chambers for 5 minutes. The mice are allowed to inhale anaerosol containing 80 mg/ml methacholine following inhalation treatmentwith DPBS vehicle (Dulbecco's Phosphate Buffered Saline), or 80 mg/mlmethacholine following inhalation treatment with test compound. Theaverage enhanced pause (Penh, lung resistance), corresponding to airflowresistance, is determined and statistically analyzed usingKruskal-Wallis one way ANOVA. In order to determine the baseline, salineaerosol (without methacholine) is also separately administered to themice.

This procedure is a model for inhalation treatment of asthma, chronicobstructive pulmonary disorder and other obstructive respiratory tractdisorders in which the effectiveness of the compounds of formulas (Ia)or (Ib) can be tested.

Test for Frequency of Micturition in Female Sprague-Dawley Rats

Ten female Sprague-Dawley rats having a mean weight of about 245-285 gare anesthetized with urethane (1.2 g/k, sc.). A midline incision isperformed to expose the bladder and a 230 catheter is inserted into thebladder dome for the measurement of intravesical pressure. A non-stoptransvesical cystometrogram, as described in J. Pharmacological.Methods, 15, pp. 157-167 (1986), is used, at a filling rate of 0.216ml/min. of saline, to access the filling and voiding characteristics ofthe bladder. Through the continuous cystometry method thus afforded,consecutive micturition can be recorded. Test compound is given atintravenous doses after the initial baseline micturition sequence isreliably measured for approximately 12 min. From these recordings, theabsolute values in maximum pressure obtained and the frequency ofmicturition is measured. A dose response curve illustrating the effectof test compound on the absolute micturition pressures in the range of1-50 mg/kg can be obtained. This procedure is a model for overactivebladder (OAB) in which the compounds of the formulas (Ia) and (Ib) canbe tested.

The following Examples illustrate numerous formulations suitable foradministering the compounds of formula (Ia) and (Ib) to treat variousconditions responsive to treatment with an anticholinergic agent. Inthese Examples, percentages are by weight unless otherwise indicated.

EXAMPLES OF PHARMACEUTICAL FORMULATIONS Example 1

Tablets per tablet Compound of formula (Ia) or (Ib), e.g. 100 mgCompound (d) or (m) or (w) lactose 140 mg corn starch 240 mgpolyvinylpyrrolidone  15 mg magnesium stearate  5 mg 500 mg

The finely ground active substance, lactose and some of the corn starchare mixed together. The mixture is screened, then moistened with asolution of polyvinylpyrrolidone in water, kneaded, wet-granulated anddried. The granules, the remaining corn starch and the magnesiumstearate are screened and mixed together. The mixture is compressed toproduce tablets of suitable shape and size.

Example 2

Tablets per tablet Compound of formula (Ia) or (Ib), e.g. 80 mg Compound(d) or (m) or (w) lactose 55 mg corn starch 190 mg  microcrystallinecellulose 35 mg polyvinylpyrrolidone 15 mg sodium-caroxymethyl starch 23mg magnesium stearate  2 mg 400 mg 

The finely ground active substance, some of the corn starch, lactose,microcrystaline cellulose and polyvinylpyrrolidone are mixed together,the mixture is screened and worked with the remaining corn starch andwater to form a granulate which is dried and screened. The sodiumcarboxymethyl starch and the magnesium stearate are added and mixed inand the mixture is compressed to form tablets of a suitable size.

Example 3

Ampule solution Compound of formula (Ia) or (Ib), e.g. 50 mg Compound(d) or (m) or (w) sodium chloride 50 mg water for inj. 5 ml

The active substance is dissolved in water at its own pH or optionallyat pH 5.5 to 6.5 and sodium chloride is added to make it isotonic. Thesolution obtained is filtered free from pyrogens and the filtrate istransferred under aseptic conditions into ampules which are thensterilized and sealed by fusion. The ampules contain 5 mg, 25 mg and 50mg of active substance.

Example 4

Metering aerosol Compound of formula (Ia) or (Ib), e.g. 0.005 Compound(d) or (m) or (w) Sorbitan trioleate 0.1 Monofluorotrichloromethane and2:3 ad 100 difluorodichioromethane

The suspension is transferred into a conventional aerosol container witha metering valve. Preferably, 50 μl of suspension are delivered perspray. The active substance may also be metered in higher doses ifdesired (e.g. 0.02% by weight).

Example 5

Solutions (in mg/100 ml) Compound of formula (Ia) or (Ib), e.g. 333.3mg  Compound (d) or (m) or (w) Formoterol fumarate 333.3 mg Benzalkonium chloride 10.0 mg EDTA 50.0 mg HCl(ln) ad pH 3.4

This solution may be prepared in the usual manner.

Example 6

Powder for inhalation Compound of formula (Ia) or (Ib), e.g. 6 μgCompound (d) or (m) or (w) Formoterol fumarate 6 μg Lactose monohydratead 25 mg

The powder for inhalation is produced in the usual way by mixing theindividual ingredients together.

Example 7

Powder for inhalation Compound of formula (Ia) or (Ib), e.g. 10 μgCompound (d) or (m) or (w) Lactose monohydrate ad 5 mg

The powder for inhalation is produced in the usual way by mixing theindividual ingredients together.

Further Formulations Obtained Analogously to Methods Known in the Art A:Inhalable Powders Example 8

Ingredients μg per capsule Compound of formula (Ia) or (Ib), e.g. 100Compound (d) or (m) or (w) budesonide 200 lactose 4700 Total 5000

Example 9

Ingredients μg per capsule Compound of formula (Ia) or (Ib), e.g. 100Compound (d) or (m) or (w) fluticasone propionate 125 lactose 4775 Total5000

Example 10

Ingredients μg per capsule Compound of formula (Ia) or (Ib), e.g. 100Compound (d) or (m) or (w) mometasone furoate × H₂O 250 lactose 4650Total 5000

Example 11

Ingredients μg per capsule Compound of formula (Ia) or (Ib), e.g. 100Compound (d) or (m) or (w) ciclesonide 250 lactose 4650 Total 5000

Example 12

Ingredients μg per capsule Compound of formula (Ia) or (Ib), e.g. 50Compound (d) or (m) or (w) budesonide 125 lactose 4825 Total 5000

Example 13

Ingredients μg per capsule Compound of formula (Ia) or (Ib), e.g. 50Compound (d) or (m) or (w) fluticasone propionate 200 lactose 4750 Total5000

Example 14

Ingredients μg per capsule Compound of formula (Ia) or (Ib), e.g. 75Compound (d) or (m) or (w) mometasone furoate × H₂O 250 lactose 4675Total 5000

Example 15

Ingredients μg per capsule Compound of formula (Ia) or (Ib), e.g. 75Compound (d) or (m) or (w) ciclesonide 250 lactose 4675 Total 5000

Example 16

Ingredients μg per capsule Compound of formula (Ia) or (Ib), e.g. 100Compound (d) or (m) or (w) ST-126 250 lactose 4650 Total 5000

Example 17

Ingredients μg per capsule Compound of formula (Ia) or (Ib), e.g. 50Compound (d) or (m) or (w) ST-126 125 lactose 4825 Total 5000

Example 18

Ingredients μg per capsule Compound of formula (Ia) or (Ib), e.g. 100Compound (d) or (m) or (w) loteprednol etabonate 200 lactose 4700 Total5000

Example 19

Ingredients μg per capsule Compound of formula (Ia) or (Ib), e.g. 100Compound (d) or (m) or (w) etiprednol dichloroacetate 200 lactose 4700Total 5000

Example 20

Ingredients μg per capsule Compound of formula (Ia) or (Ib), e.g. 100Compound (d) or (m) or (w) loteprednol etabonate 125 lactose 4775 Total5000

Example 21

Ingredients μg per capsule Compound of formula (Ia) or (Ib), e.g. 50Compound (d) or (m) or (w) etiprednol dichloroacetate 125 lactose 4825Total 5000

Example 22

Ingredients μg per capsule Compound of formula (Ia) or (Ib), e.g. 100Compound (d) or (m) or (w) loteprednol etabonate 200 Δ¹ - cortienic acidmethyl ester 200 lactose 4500 Total 5000

Example 23

Ingredients μg per capsule Compound of formula (Ia) or (Ib), e.g. 100Compound (d) or (m) or (w) loteprednol etabonate 200 Δ¹ - cortienic acid200 lactose 4500 Total 5000

Example 24

Ingredients μg per capsule Compound of formula (Ia) or (Ib), e.g. 100Compound (d) or (m) or (w) loteprednol etabonate 125 Δ¹ - cortienic acidor 125 Δ¹ - cortienic acid methyl ester lactose 4650 Total 5000

Example 25

Ingredients μg per capsule Compound of formula (Ia) or (Ib), e.g. 50Compound (d) or (m) or (w) loteprednol etabonate 125 Δ¹ - cortienic acidor 125 Δ¹ - cortienic acid methyl ester lactose 4700 Total 5000

B. Propellant-Containing Aerosols for Inhalation (wherein TO 134a is1,1,1,2-tetrafluoroethane and TG 227 is1,1,1,2,3,3,3-heptafluoropropane) Example 26 Suspension Aerosol

Ingredients % by weight Compound of formula (Ia) or (Ib), e.g. 0.050Compound (d) or (m) or (w) budesonide 0.4 soya lecithin 0.2 TG134a:TG227 (2:3) to 100

Example 27 Suspension Aerosol

Ingredients % by weight Compound of formula (Ia) or (Ib), e.g. 0.020Compound (d) or (m) or (w) fluticasone propionate 0.3 isopropylmyristate 0.1 TG 227 to 100

Example 28 Suspension Aerosol

Ingredients % by weight Compound of formula (Ia) or (Ib), e.g. 0.020Compound (d) or (m) or (w) mometasone furoate × H₂O 0.6 isopropylmyristate 0.1 TG 227 to 100

Example 29 Suspension Aerosol

Ingredients % by weight Compound of formula (Ia) or (Ib), e.g. 0.020Compound (d) or (m) or (w) ciclesonide 0.4 isopropyl myristate 0.1 TG134a:TG227 (2:3) to 100

Example 30 Suspension Aerosol

Ingredients % by weight Compound of formula (Ia) or (Ib), e.g. 0.039Compound (d) or (m) or (w) ciclesonide 0.4 absolute ethanol 0.5isopropyl myristate 0.1 TG 134a:TG227 (2:3) to 100

Example 31 Solution Aerosol

Ingredients % by weight Compound of formula (Ia) or (Ib), e.g. 0.039Compound (d) or (m) or (w) fluticasone propionate 0.2 absolute ethanol0.5 isopropyl myristate 0.1 TG 134a:TG227 (2:3) to 100

Example 32 Solution Aerosol

Ingredients % by weight Compound of formula (Ia) or (Ib), e.g. 0.039Compound (d) or (m) or (w) mometasone furoate × H₂O 0.6 absolute ethanol0.5 isopropyl myristate 0.1 TG 134a:TG227 (2:3) to 100

Example 33 Solution Aerosol

Ingredients % by weight Compound of formula (Ia) or (Ib), e.g. 0.039Compound (d) or (m) or (w) ciclesonide 0.4 absolute ethanol 0.5isopropyl myristate 0.1 TG 134a:TG227 (2:3) to 100

Example 34 Solution Aerosol

Ingredients % by weight Compound of formula (Ia) or (Ib), e.g. 0.039Compound (d) or (m) or (w) ST-126 0.6 absolute ethanol 0.5 isopropylmyristate 0.1 TG 134a:TG227 (2:3) to 100

Example 35 Solution Aerosol

Ingredients % by weight Compound of formula (Ia) or (Ib), e.g. 0.039Compound (d) or (m) or (w) ST-126 0.4 absolute ethanol 0.5 isopropylmyristate 0.1 TG 134a:TG227 (2:3) to 100

Example 36

Ingredients % by weight Compound of formula (Ia) or (Ib), e.g. 0.05Compound (d) or (m) or (w) loteprednol etabonate 0.4 soya lecithin 0.2TG 134a:TG227 (2:3) to 100

Example 37

Ingredients % by weight Compound of formula (Ia) or (Ib), e.g. 0.020Compound (d) or (m) or (w) loteprednol etabonate 0.3 isopropyl myristate0.1 TG 227 to 100

Example 38

Ingredients % by weight Compound of formula (Ia) or (Ib), e.g. 0.020Compound (d) or (m) or (w) etiprednol dichloracetate 0.4 isopropylmyristate 0.1 TG 227 to 100

Example 39

Ingredients % by weight Compound of formula (Ia) or (Ib), e.g. 0.020Compound (d) or (m) or (w) loteprednol etabonate 0.4 isopropyl myristate0.1 TG 134a:TG227 (2:3) to 100

Example 40

Ingredients % by weight Compound of formula (Ia) or (Ib), e.g. 0.039Compound (d) or (m) or (w) loteprednol etabonate 0.4 absolute ethanol0.5 isopropyl myristate 0.1 TG 134a:TG227 (2:3) to 100

Example 41

Ingredients % by weight Compound of formula (Ia) or (Ib), e.g. 0.05Compound (d) or (m) or (w) loteprednol etabonate 0.4 Δ¹- cortienic acidor Δ¹- cortienic acid 0.4 methyl ester soya lecithin 0.2 TG134a:TG227(2:3) to 100

Example 42

Ingredients % by weight Compound of formula (Ia) or (Ib), e.g. 0.02Compound (d) or (m) or (w) loteprednol etabonate 0.3 Δ¹- cortienic acidor Δ¹- cortienic acid 0.3 methyl ester isopropyl myristate 0.1 TG227 to100

Example 43

Ingredients % by weight Compound of formula (Ia) or (Ib), e.g. 0.04Compound (d) or (m) or (w) loteprednol etabonate 0.4 Δ¹- cortienic acidor Δ¹- cortienic acid 0.4 methyl ester isopropyl myristate 0.1 TG 227 to100

Example 44

Ingredients % by weight Compound of formula (Ia) or (Ib), e.g. 0.02Compound (d) or (m) or (w) loteprednol etabonate 0.4 Δ¹- cortienic acidor Δ¹- cortienic acid 0.4 methyl ester isopropyl myristate 0.1TG134a:TG227 (2:3) to 100

Example 45

Ingredients % by weight Compound of formula (Ia) or (Ib), e.g. 0.039Compound (d) or (m) or (w) loteprednol etabonate 0.4 Δ¹- cortienic acidor Δ¹- cortienic acid 0.4 methyl ester absolute ethanol 0.5 isopropylmyristate 0.1 TG134a:TG227 (2:3) to 100

C. Ophthalmic Formulations Example 46

EYE DROPS Compound of formula (Ia) or (Ib), e.g. 0.05% w/v Compound (d)or (m) or (w) Tween 80 2.5% w/v Ethanol 0.75% w/v Benzalkonium chloride0.02% w/v Phenyl ethanol 0.25% w/v Sodium chloride 0.60% w/v Water forinjection q.s. 100 volumes

Example 47

EYE DROPS Compound of formula (Ia) or (Ib), e.g. 0.04% w/v Compound (d)or (m) or (w) Tween 80 2.5% w/v Ethanol 0.75% w/v Benzalkonium chloride0.02% w/v Phenyl ethanol 0.25% w/v Sodium chloride 0.60% w/v Water forinjection q.s. 100 volumes

Example 48

EYE DROPS Compound of formula (Ia) or (Ib), e.g. 0.035% w/v Compound (d)or (m) or (w) Povidone 0.6% w/v Benzalkonium chloride 0.02% w/v Sodiumedetate U.S.P. 0.10% w/v Glycerin U.S.P. 2.5% w/v Tyloxapol U.S.P. 3.0%w/v Sodium chloride 0.3% w/v Sodium γ-aminobutyrate 1.0% w/v Steriledistilled water q.s. 100 volumes

The ingredients listed above are combined, then the is checked and, ifnecessary, adjusted to 5.0-5.5 by basifying with sodium hydroxide oracidifying with hydrochloric acid.

Yet other compositions of the invention can be conveniently formulatedusing known techniques.

While this description has been couched in terms of various preferred orexemplary embodiments, the skilled artisan will appreciate that variousmodifications, substitutions, omissions and changes may be made withoutdeparting from the spirit thereof Accordingly, it is intended that thescope of the foregoing be limited only by the broadest statements hereinand by the scope of the following claims, including equivalents thereof.

What is claimed is:
 1. A method for eliciting an anticholinergicresponse in a subject in need of same, comprising administering to saidsubject an anticholinergically effective amount of a compound having theformula:

wherein R is methyl or ethyl; and wherein each asterisk marks a chiralcenter; said compound having the R, S or RS stereoisomeric configurationat each chiral center unless specified otherwise, or being a mixturethereof.
 2. A method as claimed in claim 1 for reducing or inhibitingthe development of, or alleviating the symptoms of, chronic obstructivepulmonary disease or asthma in a subject in need thereof, comprisingadministering to said subject an anticholinergically effective amount ofa compound having the formula:

wherein R is methyl or ethyl; and wherein each asterisk marks a chiralcenter; said compound having the R, S or RS stereoisomeric configurationat each chiral center unless specified otherwise, or being a mixturethereof.
 3. A method as claimed in claim 1 for inducing mydriasis in theeye(s) of a subject in need thereof, comprising topically applying tothe eye(s) of said subject a mydriatically effective amount of acompound having the formula:

wherein R is methyl or ethyl; and wherein each asterisk marks a chiralcenter; said compound having the R, S or RS stereoisomeric configurationat each chiral center unless specified otherwise, or being a mixturethereof.
 4. A method as claimed in claim 1 for alleviating the symptomsof overactive bladder in a subject in need thereof, comprisingadministering to said subject an anticholinergically effective amount ofa compound having the formula:

wherein R is methyl or ethyl; and wherein each asterisk marks a chiralcenter; said compound having the R, S or RS stereoisomeric configurationat each chiral center unless specified otherwise, or being a mixturethereof.
 5. A method as claimed in claim 1 for eliciting anantiperspirant effect in a subject in need thereof, comprising topicallyapplying to said subject an antiperspirant effective amount of acompound having the formula:

wherein R is methyl or ethyl; and wherein each asterisk marks a chiralcenter; said compound having the R, S or RS stereoisomeric configurationat each chiral center unless specified otherwise, or being a mixturethereof.
 6. The method as claimed in claim 1, wherein the compound is:3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide;3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,3′S) (2R,3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,1′R,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,1′S,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,1′R,3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; or (2R,1′S,3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide.
 7. The method as claimed in claim 1, wherein the compound is:(a)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (b)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (c) (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; or (d) (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide.
 8. A method for eliciting an anticholinergic response selectedfrom the group consisting of a mydriatic response and an antiperspirantresponse in a subject in need of same, comprising administering to saidsubject's eye(s) or skin, respectively, an anticholinergically effectiveamount of a compound having the formula:

wherein R is methyl or ethyl; and wherein each asterisk marks a chiralcenter; said compound having the R, S or RS stereoisomeric configurationat each chiral center unless specified otherwise, or being a mixturethereof.
 9. The method as claimed in claim 2, wherein the compound is:3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide;3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,1′R,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,1′S,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,1′R,3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; or (2R,1′S,3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide.
 10. The method as claimed in claim 2, wherein the compound is:(a)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (b)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (c) (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; or (d) (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide.
 11. The method as claimed in claim 3, wherein the compound is:3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide;3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,1′R, 3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,1S,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,1′R,3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; or (2R,1′S,3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide.
 12. The method as claimed in claim 4, wherein the compound is:(a)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (b)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (c) (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; or (d) (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide.
 13. The method as claimed in claim 4, wherein the compound is:3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide;3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,1′R,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,1′S,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,1′R,3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; or (2R,1′S,3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide.
 14. The method as claimed in claim 4, wherein the compound is:(a)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (b)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (c) (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; or (d) (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide.
 15. The method as claimed in claim 5, wherein the compound is:3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide;3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,1′R,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,1′S 3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,1′R,3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; or (2R,1S,3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide.
 16. The method as claimed in claim 5, wherein the compound is:(a)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (b)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (c) (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; or (d) (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide.
 17. The method as claimed in claim 8, wherein the compound is:3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide;3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,1′R,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,1′S,3′S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (2R,1′R,3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; or (2R,1′S,3′R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide.
 18. The method as claimed in claim 8, wherein the compound is:(a)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (b)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; (c) (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-methylpyrrolidiniumbromide; or (d) (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-methylpyrrolidiniumbromide.